ILT7 binding molecules and methods of using the same

ABSTRACT

The present invention is directed to ILT7 binding molecules, e.g., anti-ILT7 antibodies, and methods for treating or preventing conditions and diseases associated with ILT7-expressing cells such as autoimmune diseases.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a U.S. National Stage entry filed under 35 U.S.C. §371 of International Application PCT/US17/21616 filed on Mar. 9, 2017,and claims the benefit of and priority to U.S. Provisional Applicationfor Patent Ser. No. 62/306,125 filed Mar. 10, 2016, the entire contentsof each of which are incorporated by reference herein in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing in an ASCIItext file (Name 054493-506N01US_SL.txt; Size: 148,542 bytes; and Date ofCreation: Dec. 17, 2020) is incorporated herein by reference in itsentirety.

BACKGROUND OF THE INVENTION

Plasmacytoid dendritic cells (pDCs) are a distinct population ofdendritic cells (DC) in the peripheral blood and secondary lymphoidorgans that make up only about 0.1 to 0.5% of peripheral bloodmononuclear cells (PBMC). However, these cells are particularlyimportant regulators of the immune system because they are the mainsource of Type-I interferon (IFN). Type-I IFNs promote the function ofNK cells, B cells, T cells, and myeloid dendritic cells. These IFNs areimportant in initial immune responses and have antiviral and antitumoractivity. However, pDCs and Type-I IFNs are also thought to play a rolein the development of autoimmune diseases such as systemic lupuserythematosus, chronic rheumatism, and psoriasis. Therefore,understanding how to regulate the molecular pathways involved in IFNrelease is useful for controlling immune responses and treating andpreventing disease.

pDCs release IFN in response to nucleic acids that are sensed byToll-like receptors (TLRs) TLR7 and TLR9 expressed on the surface of thepDCs. The TLR-induced response is regulated by receptors containingimmunoreceptor tyrosine-based activation motifs (ITAMs).Immunoglobulin-Like transcript-7 (ILT7), also called LIRA4, LILRA4, orCD85g, is one such receptor.

ILT7 is a member of the immunoglobulin-like transcript (ILT) orleukocyte immunoglobulin-like receptor (LIR) gene family. ILT7 isselectively expressed on the surface of human plasmacytoid dendritecells (pDCs) and is not on myeloid dendritic cells or other peripheralblood leukocytes. Cao et al., J. Exp. Medicine 6:1399-1405 (2006). ILT7contains four immunoglobulin-like extracellular domains and atransmembrane domain. The extracellular portion is important forinteracting with the ILT7 ligand, bone marrow stromal cell antigen 2(BST2), and the transmembrane domain of ILT7 contains a positivelycharged residue that allows it to complex with FcεRIγ. It has beenpostulated that the BST2-ILT7 interaction negatively regulates theinnate immune function of pDCs, potentially as a mechanism of negativefeed back. In addition, in vitro antibody cross-linking of ILT7 has beenshown to negatively regulate the production of IFN-alpha and TNF-alphaby pDCs. Therefore, antibodies and other ILT7 binding molecules that areuseful for neutralizing ILT7 and regulating pDC activity and IFN releaseare needed, for example, for treating and preventing diseases such asautoimmune diseases.

FIELD OF THE INVENTION

The invention relates to ILT7 binding molecules, e.g., anti-ILT7antibodies and antigen-binding fragments, variants, or derivativesthereof, methods of using the antibodies and fragments, and methods fortreating or preventing autoimmune diseases and conditions associatedwith ILT7-expressing cells.

BRIEF SUMMARY OF THE INVENTION

Provided herein are ILT7 binding molecules, e.g., anti-ILT7 antibodiesand antigen-binding fragments thereof.

In one instance, an isolated ILT7 binding protein is an ILT7 bindingprotein that can bind to the same ILT7 epitope as an antibody comprisinga heavy chain variable region (VH) of SEQ ID NO:202 and a light chainvariable region (VL) of SEQ ID NO:207.

In one instance, an isolated ILT7 binding protein is an ILT7 bindingprotein that competitively inhibits the binding to ILT7 of an antibodycomprising a VH of SEQ ID NO:202 and a VL of SEQ ID NO:207

In one instance, an isolated ILT7 binding protein is an ILT7 bindingprotein comprising Complementarity-Determining Regions (CDRs) HCDR1,HDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprising the sequences of SEQ IDNOs: 203, 204, 205, 208, 209, and 210, respectively.

In one instance, the ILT7 binding protein comprises a VH at least 85%,90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:202 and/or a VL atleast 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:207.

In one instance, the ILT7 binding protein comprises a VH comprising SEQID NO:202 and a VL comprising SEQ ID NO:207.

In one instance, an isolated ILT7 binding protein is an ILT7 bindingprotein comprising a VH comprising SEQ ID NO:202.

In one instance, an isolated ILT7 binding protein is an ILT7 bindingprotein comprising a VH comprising SEQ ID NO:207.

In one instance, an isolated ILT7 binding protein is an ILT7 bindingprotein that can bind to the same ILT7 epitope as an antibody comprisinga VH and a VL selected from the group consisting of SEQ ID NO:12 and SEQID NO:17, respectively SEQ ID NO:22 and SEQ ID NO:27, respectively; SEQID NO:32 and SEQ ID NO:37, respectively; SEQ ID NO:42 and SEQ ID NO:47,respectively; SEQ ID NO:52 and SEQ ID NO:57, respectively; SEQ ID NO:62and SEQ ID NO:67, respectively; SEQ ID NO:72 and SEQ ID NO:77,respectively; SEQ ID NO:82 and SEQ ID NO:87, respectively; SEQ ID NO:92and SEQ ID NO:97, respectively; SEQ ID NO:102 and SEQ ID NO:107,respectively; SEQ ID NO:112 and SEQ ID NO:117, respectively; SEQ IDNO:122 and SEQ ID NO:127, respectively; SEQ ID NO:132 and SEQ ID NO:137,respectively; SEQ ID NO:142 and SEQ ID NO:147, respectively; SEQ IDNO:152 and SEQ ID NO:157, respectively; SEQ ID NO:162 and SEQ ID NO:167,respectively; SEQ ID NO:172 and SEQ ID NO:177, respectively; SEQ IDNO:182 and SEQ ID NO:187, respectively; SEQ ID NO:192 and SEQ ID NO:197,respectively; SEQ ID NO:212 and SEQ ID NO:217, respectively; SEQ IDNO:222 and SEQ ID NO:227, respectively; SEQ ID NO:232 and SEQ ID NO:237,respectively; and SEQ ID NO:242 and SEQ ID NO:247, respectively.

In one instance, an isolated ILT7 binding protein is an ILT7 bindingprotein that competitively inhibits the binding to ILT7 of an antibodycomprising a VH and VL selected from the group consisting of SEQ IDNO:12 and SEQ ID NO:17, respectively SEQ ID NO:22 and SEQ ID NO:27,respectively; SEQ ID NO:32 and SEQ ID NO:37, respectively; SEQ ID NO:42and SEQ ID NO:47, respectively; SEQ ID NO:52 and SEQ ID NO:57,respectively; SEQ ID NO:62 and SEQ ID NO:67, respectively; SEQ ID NO:72and SEQ ID NO:77, respectively; SEQ ID NO:82 and SEQ ID NO:87,respectively; SEQ ID NO:92 and SEQ ID NO:97, respectively; SEQ ID NO:102and SEQ ID NO:107, respectively; SEQ ID NO:112 and SEQ ID NO:117,respectively; SEQ ID NO:122 and SEQ ID NO:127, respectively; SEQ IDNO:132 and SEQ ID NO:137, respectively; SEQ ID NO:142 and SEQ ID NO:147,respectively; SEQ ID NO:152 and SEQ ID NO:157, respectively; SEQ IDNO:162 and SEQ ID NO:167, respectively; SEQ ID NO:172 and SEQ ID NO:177,respectively; SEQ ID NO:182 and SEQ ID NO:187, respectively; SEQ IDNO:192 and SEQ ID NO:197, respectively; SEQ ID NO:212 and SEQ ID NO:217,respectively; SEQ ID NO:222 and SEQ ID NO:227, respectively; SEQ IDNO:232 and SEQ ID NO:237, respectively; and SEQ ID NO:242 and SEQ IDNO:247, respectively.

In one instance, an isolated ILT7 binding protein is an ILT7 bindingprotein comprising CDRs: HCDR1, HDR2, HCDR3, LCDR1, LCDR2, and LCDR3selected from the group consisting of SEQ ID NOs: 13, 14, 15, 18, 19,and 20, respectively; SEQ ID NOs: 23, 24, 25, 28, 29, and 30,respectively; SEQ ID NOs: 33, 34, 35, 38, 39, and 40, respectively; SEQID NOs: 103, 104, 105, 108, 109, and 110, respectively; SEQ ID NOs: 213,214, 215, 218, 219, and 220, respectively; SEQ ID NOs: 223, 224, 225,228, 229, and 230, respectively; SEQ ID NOs: 233, 234, 235, 238, 239,and 240, respectively; and SEQ ID NOs: 243, 244, 245, 248, 249, and 250;respectively.

In one instance, the ILT7 binding protein comprises a VH and a VL atleast 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to: SEQ ID NO:12 andSEQ ID NO:17, respectively; SEQ ID NO:22 and SEQ ID NO:27, respectively;SEQ ID NO:32 and SEQ ID NO:37, respectively; SEQ ID NO:42 and SEQ IDNO:47, respectively; SEQ ID NO:52 and SEQ ID NO:57, respectively; SEQ IDNO:62 and SEQ ID NO:67, respectively; SEQ ID NO:72 and SEQ ID NO:77,respectively; SEQ ID NO:82 and SEQ ID NO:87, respectively; SEQ ID NO:92and SEQ ID NO:97, respectively; SEQ ID NO:102 and SEQ ID NO:107,respectively; SEQ ID NO:112 and SEQ ID NO:117, respectively; SEQ IDNO:122 and SEQ ID NO:127, respectively; SEQ ID NO:132 and SEQ ID NO:137,respectively; SEQ ID NO:142 and SEQ ID NO:147, respectively; SEQ IDNO:152 and SEQ ID NO:157, respectively; SEQ ID NO:162 and SEQ ID NO:167,respectively; SEQ ID NO:172 and SEQ ID NO:177, respectively; SEQ IDNO:182 and SEQ ID NO:187, respectively; SEQ ID NO:192 and SEQ ID NO:197,respectively; SEQ ID NO:212 and SEQ ID NO:217, respectively; SEQ IDNO:222 and SEQ ID NO:227, respectively; SEQ ID NO:232 and SEQ ID NO:237,respectively; or SEQ ID NO:242 and SEQ ID NO:247, respectively.

In one instance, the VH and VL comprise SEQ ID NO:12 and SEQ ID NO:17,respectively; SEQ ID NO:22 and SEQ ID NO:27, respectively; SEQ ID NO:32and SEQ ID NO:37, respectively; SEQ ID NO:42 and SEQ ID NO:47,respectively; SEQ ID NO:52 and SEQ ID NO:57, respectively; SEQ ID NO:62and SEQ ID NO:67, respectively; SEQ ID NO:72 and SEQ ID NO:77,respectively; SEQ ID NO:82 and SEQ ID NO:87, respectively; SEQ ID NO:92and SEQ ID NO:97, respectively; SEQ ID NO:102 and SEQ ID NO:107,respectively; SEQ ID NO:112 and SEQ ID NO:117, respectively; SEQ IDNO:122 and SEQ ID NO:127, respectively; SEQ ID NO:132 and SEQ ID NO:137,respectively; SEQ ID NO:142 and SEQ ID NO:147, respectively; SEQ IDNO:152 and SEQ ID NO:157, respectively; SEQ ID NO:162 and SEQ ID NO:167,respectively; SEQ ID NO:172 and SEQ ID NO:177, respectively; SEQ IDNO:182 and SEQ ID NO:187, respectively; SEQ ID NO:192 and SEQ ID NO:197,respectively; SEQ ID NO:212 and SEQ ID NO:217, respectively; SEQ IDNO:222 and SEQ ID NO:227, respectively; SEQ ID NO:232 and SEQ ID NO:237,respectively; or SEQ ID NO:242 and SEQ ID NO:247, respectively.

In one instance, an isolated ILT7 binding protein comprises a VHcomprising SEQ ID NO: 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122,132, 142, 152, 162, 172, 182, 192, 212, 222, 232, or 242.

In one instance, an isolated ILT7 binding protein comprises a VLcomprising SEQ ID NO: 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127,137, 147, 157, 167, 177, 187, 197, 217, 227, 237, or 247.

In one instance, the ILT7 binding protein comprises an antibody orantigen-binding fragment thereof. In one instance, the antibody orantigen-binding fragment thereof is afucosylated.

In one instance, the ILT7 binding protein binds to the Ig1 region ofILT7. In one instance, the ILT7 binding protein binds to the Ig2 regionof ILT7.

In one instance, the ILT7 binding protein binds to human and cynomolgusILT7.

In one instance, the ILT7 binding protein suppresses interferon (IFN)alpha release from peripheral blood mononuclear cells (PBMCs). In oneinstance, the ILT7 binding protein has ADCC activity againstplasmacytoid dendritic cells (pDCs) in PMBCs.

In one instance, the ILT7 binding protein comprises a murine, human,chimeric, humanized, or resurfaced antibody or antigen-binding fragmentthereof.

In one instance, the ILT7 binding protein comprises an antibody, Fab,Fab′, F(ab′)2, Fd, single chain Fv or scFv, disulfide linked Fv, V-NARdomain, IgNar, intrabody, IgGΔCH2, minibody, F(ab′)3, tetrabody,triabody, diabody, single-domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2,or scFv-Fc.

In one instance, the ILT7 binding protein comprises a monoclonalantibody or an antigen binding fragment thereof.

In one instance, the ILT7 binding protein comprises a heavy chainimmunoglobulin constant domain selected from the group consisting of:(a) an IgA constant domain; (b) an IgD constant domain; (c) an IgEconstant domain; (d) an IgG1 constant domain; (e) an IgG2 constantdomain; (f) an IgG3 constant domain; (g) an IgG4 constant domain; and(h) an IgM constant domain.

In one instance, the ILT7 binding protein comprises a light chainimmunoglobulin constant domain selected from the group consisting of:(a) an Ig kappa constant domain; and (b) an Ig lambda constant domain.

In one instance, the ILT7 binding protein comprises a human IgG1constant domain and a human lambda constant domain.

In one instance, provided herein is a host cell producing the ILT7binding molecule.

In one instance, provided herein is an isolated polynucleotidecomprising a nucleic acid encoding a VH, wherein the VH comprises anamino acid sequence at least 85%, 90%, 95% identical, or identical tothe VH of SEQ ID NO: 202, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112,122, 132, 142, 152, 162, 172, 182, 192, 212, 222, 232, or 242. In oneinstance, the polynucleotide comprises a sequence at least 85%, 90%, 95%identical, or identical to SEQ ID NO:201, 11, 21, 31, 41, 51, 61, 71,81, 91, 101, 111, 121, 131, 141, 151, 161, 171, 181, 191, 211, 221, 231,or 241.

In one instance, provided herein is an isolated polynucletide comprisinga nucleic acid encoding a VL, wherein the VL comprises an amino acidsequence at least 85%, 90%, 95% identical, or identical to the VL of207, 17, 27, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, 147, 157, 167,177, 187, 197, 217, 227, 237, or 247. In one instance, thepolynucleotide comprises a sequence at least 85%, 90%, 95% identical, oridentical to SEQ ID NO: 206, 16, 26, 36, 46, 56, 66, 76, 86, 96, 106,116, 126, 136, 146, 156, 166, 176, 186, 196, 216, 226, 236, or 246.

In one instance, the nucleic acid is operably linked to a controlsequence. In one instance, an antibody or antigen-binding fragmentthereof comprising the VH or the VL encoded by the nucleic acid canspecifically bind to ILT7.

In one instance, a polynucleotide encodes an ILT7 binding moleculeprovided herein.

In one instance, provided herein is a vector comprising thepolynucleotide.

In one instance, provided herein is a polypeptide encoded by thepolynucleotide.

In one instance, provided herein is a host cell transformed with apolynucleotide provided herein (e.g., a polynucleotide comprising anucleic acid encoding a VH and a polynucleotide comprising a nucleicacid encoding a VL).

In one instance, provided herein is a host cell comprising apolynucleotide provided herein (e.g., a polynucleotide comprising anucleic acid encoding a VH and a polynucleotide comprising a nucleicacid encoding a VL), a vector provided herein, or a polypeptide providedherein. In one instance, the host cell is a mammalian host cell. In oneinstance, the host cell is a NS0 murine myeloma cell, a PER.C6® humancell, or a Chinese hamster ovary (CHO) cells. In one instance, the hostcell lacks the enzyme α-1,6-fucosyltransferase.

In one instance, provided herein is a method of producing an anti-ILT7binding molecule, comprising culturing a host cell provided herein andrecovering said binding molecule. In one instance, provided herein is ananti-ILT7 binding molecule, produced by the method.

In one instance, provided herein is a method for detecting ILT7expression in a sample comprising (a) contacting the sample with an ILT7binding molecule provided herein and (b) detecting binding of thebinding molecule in the sample.

In one instance, provided herein is a method for detecting plasmacytoiddendritic cells comprising (a) contacting a sample containing cells withan ILT7 binding molecule provided herein and (b) detecting binding ofthe binding molecule in the sample.

In one instance, provided herein is a pharmaceutical compositioncomprising (a) an ILT7 binding molecule provided herein, apolynucleotide provided herein, a vector provided herein, a polypeptideprovided herein, or a host cell provided herein and (b) a carrier.

In one instance, provided herein is a method for decreasing IFN-alpharelease from a plasmacytoid dendritic cell, comprising contacting aplasmacytoid dendritic cell with an ILT7 binding molecule providedherein, a polynucleotide provided herein, a vector provided herein, apolypeptide provided herein, a host cell provided herein, or apharmaceutical composition provided herein.

In one instance, provided herein is a method for treating a humansubject with an autoimmune disease comprising administering to thesubject an effective amount of an ILT7 binding molecule provided herein,a polynucleotide provided herein, a vector provided herein, apolypeptide provided herein, a host cell provided herein, or apharmaceutical composition provided herein.

In one instance, provided herein is a method for preventing anautoimmune disease in a human subject comprising administering to thesubject an effective amount of an ILT7 binding molecule provided herein,a polynucleotide provided herein, a vector provided herein, apolypeptide provided herein, a host cell provided herein, or apharmaceutical composition provided herein. In one instance, theautoimmune disease is systemic lupus erythematosus. In one instance, theautoimmune disease is chronic rheumatism.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIGS. 1A and 1B: show the variable heavy chain (1A) and variable lightchain (1B) sequence alignments of the SBI28 (#28), 10D10, and 7C7antibodies. Shading indicates CDR sequences. Open boxes representmutations introduced in 10D10 to generate 7C7. FIG. 1A discloses SEQ IDNOS 2, 293, and 12 and FIG. 1B discloses SEQ ID NOS 7, 294, and 17,respectively, in order of appearance.

FIG. 2: shows the binding of ILT7 antibodies and negative controlantibody (R437) to CT-550 cells expressing human ILT7 as determined byflow cytometry. SBI33 refers to the anti-ILT7 antibody ILT7#33 asprovided in U.S. Published Application No. 2009/0280128.

FIG. 3: shows the binding of ILT7 antibodies and negative controlantibody (R437) to CT-125 cells expressing cynomolgus ILT7 as determinedby flow cytometry.

FIG. 4: shows the ADCC potency of ILT7 antibodies and negative controlantibody (R437) against human ILT7-expressing cells.

FIG. 5: shows the ADCC potency of ILT7 antibodies and negative controlantibody (R437) against cynomolgus ILT7-expressing cells.

FIGS. 6A and 6B: show the binding of ILT7 antibodies and negativecontrol antibody (R437) to plasmacytoid dendritic cells (pDCs) inperipheral blood mononuclear cells (PBMCs).

FIG. 7: shows the binding of afucosylated ILT7 antibodies and theirparent antibodies to CT-550 cells expressing human (left panel) andcynomolgus (right panel) ILT7 as determined by flow cytometry.

FIG. 8: shows the ADCC potency of afucosylated ILT7 antibodies and theirparent antibodies against human (left panel) and cynomolgus (rightpanel) ILT7-expressing cells.

FIGS. 9A and 9B: show the variable heavy chain (9A) and variable lightchain (9B) sequence alignments of seven ILT70080 variants. The closestgermline sequences (IGHV1-69*1 and IGLV3-21*01) are also shown in thealignments. FIG. 9A discloses SEQ ID NOS 22, 32, 42, 52, 62, 72, 82, 92,and 295 and FIG. 9B discloses SEQ ID NOS 27, 37, 47, 57, 67, 77, 87, 97,and 296, respectively, in order of appearance.

FIGS. 10A and 10B: show the variable heavy chain (10A) and variablelight chain (10B) sequence alignments of nine ILT70083 variants. Theclosest germline sequences (IGHV3-23*01 and IGLV1-51*01) are also shownin the alignments. FIG. 10A discloses SEQ ID NOS 102, 112, 122, 132,142, 152, 162, 172, 182, 192, and 297 and FIG. 10B discloses SEQ ID NOS107, 117, 127, 137, 147, 157, 167, 177, 187, 197, and 298, respectively,in order of appearance.

FIG. 11: shows the binding of ILT70080 variants to cells expressinghuman ILT7 (CT-550; top panel) and cells expressing cynomolgus ILT7(CT-125; bottom panel).

FIG. 12: shows the binding of ILT70083 variants to cells expressinghuman ILT7 (top panel) or cynomolgus ILT7 (bottom panel).

FIG. 13: shows the ADCC potency of ILT70080 variant antibodies againsthuman ILT7-expressing cells.

FIG. 14: shows the ADCC potency of ILT70083 variant antibodies againsthuman ILT7-expressing cells.

FIG. 15: shows the binding of afucosylated ILT70080.6 and ILT70083antibodies to human (left panel) and cynomolgus (right panel)ILT7-expressing cells.

FIG. 16: shows the ADCC activity of afucosylated ILT70080.6 and ILT70083antibodies on human (left panel) and cynomolgus (right panel)ILT7-expressing cells.

FIG. 17: shows the cytotoxicity (left) and IFN-α secretion (right) ofhuman PBMCs exposed to afucosylated ILT70080.6 and ILT70083 antibodies.

FIG. 18: shows binding of afucosylated ILT70137 to cells expressinghuman ILT7 (left panel) or cynomolgus ILT7 (right panel). The circlesdenote afucosylated ILT70137, and the triangles denote control.

FIG. 19: shows ADCC activity of afucosylated ILT70137 on cellsexpressing human ILT7 (left panel) or cynomolgus ILT7 (right panel). Thetriangles denote afucosylated ILT70137, and the circles denote control.

FIG. 20: shows ADCC activity of of afucosylated ILT70137 by measuringthe inhibition of IFN-alpha production as an indirect assessment of theability of the antibody to induce ADCC of peripheral blood mononuclearcells (PBMCs) in vitro.

FIG. 21: shows binding of afucosylated ILT70137 to human primaryplasmacytoid dendritic cells (pDCs).

FIG. 22: shows pDC depletion in cynomolgus monkeys treated withafucosylated 7C7 or afucosylated ILT70137. Arrows at the bottom of thegraph indicate time points of antibody administration.

FIG. 23: shows IFNα production after treatment with afucosylated 7C7 orafucosylated ILT70137. Arrows at the bottom of the graph indicate timepoints of antibody administration.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity; for example, “an anti-ILT7 antibody” is understood torepresent one or more anti-ILT7 antibodies. As such, the terms “a” (or“an”), “one or more,” and “at least one” can be used interchangeablyherein.

As used herein, the term “polypeptide” is intended to encompass asingular “polypeptide” as well as plural “polypeptides,” and refers to amolecule composed of monomers (amino acids) linearly linked by amidebonds (also known as peptide bonds). The term “polypeptide” refers toany chain or chains of two or more amino acids, and does not refer to aspecific length of the product. Thus, peptides, dipeptides, tripeptides,oligopeptides, “protein,” “amino acid chain,” or any other term used torefer to a chain or chains of two or more amino acids, are includedwithin the definition of “polypeptide,” and the term “polypeptide” canbe used instead of, or interchangeably with any of these terms. The term“polypeptide” is also intended to refer to the products ofpost-expression modifications of the polypeptide, including withoutlimitation glycosylation, acetylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, or modification by non-naturally occurring amino acids. Apolypeptide can be derived from a natural biological source or producedby recombinant technology, but is not necessarily translated from adesignated nucleic acid sequence. It can be generated in any manner,including by chemical synthesis.

A polypeptide of the invention can be of a size of about 3 or more, 5 ormore, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 ormore, 200 or more, 500 or more, 1,000 or more, or 2,000 or more aminoacids. Polypeptides can have a defined three-dimensional structure,although they do not necessarily have such structure. Polypeptides witha defined three-dimensional structure are referred to as folded, andpolypeptides that do not possess a defined three-dimensional structure,but rather can adopt a large number of different conformations, arereferred to as unfolded. As used herein, the term glycoprotein refers toa protein coupled to at least one carbohydrate moiety that is attachedto the protein via an oxygen-containing or a nitrogen-containing sidechain of an amino acid residue, e.g., a serine residue or an asparagineresidue.

By an “isolated” polypeptide or a fragment, variant, or derivativethereof is intended a polypeptide that is not in its natural milieu. Noparticular level of purification is required. For example, an isolatedpolypeptide can be removed from its native or natural environment.Recombinantly produced polypeptides and proteins expressed in host cellsare considered isolated for purpose of the invention, as are native orrecombinant polypeptides that have been separated, fractionated, orpartially or substantially purified by any suitable technique.

Also included as polypeptides of the present invention are fragments,derivatives, analogs, or variants of the foregoing polypeptides, and anycombination thereof. The terms “fragment,” “variant,” “derivative,” and“analog” when referring to anti-ILT7 antibodies or antibody polypeptidesof the present invention include any polypeptides that retain at leastsome of the antigen-binding properties of the corresponding antibody orantibody polypeptide of the invention. Fragments of polypeptides of thepresent invention include proteolytic fragments, as well as deletionfragments, in addition to specific antibody fragments discussedelsewhere herein. Variants of anti-ILT7 antibodies and antibodypolypeptides of the present invention include fragments as describedabove, and also polypeptides with altered amino acid sequences due toamino acid substitutions, deletions, or insertions. Variants can occurnaturally or be non-naturally occurring. Non-naturally occurringvariants can be produced using art-known mutagenesis techniques. Variantpolypeptides can comprise conservative or non-conservative amino acidsubstitutions, deletions, or additions. Variant polypeptides can also bereferred to herein as “polypeptide analogs.” As used herein a“derivative” of an anti-ILT7 antibody or antibody polypeptide refers toa subject polypeptide having one or more residues chemically derivatizedby reaction of a functional side group. Also included as “derivatives”are those peptides that contain one or more naturally occurring aminoacid derivatives of the twenty standard amino acids. For example,4-hydroxyproline can be substituted for proline; 5-hydroxylysine can besubstituted for lysine; 3-methylhistidine can be substituted forhistidine; homoserine can be substituted for serine; and ornithine canbe substituted for lysine. Derivatives of anti-ILT7 antibodies andantibody polypeptides of the present invention, can include polypeptidesthat have been altered so as to exhibit additional features not found onthe reference antibody or antibody polypeptide of the invention.

The term “polynucleotide” is intended to encompass a singular nucleicacid as well as plural nucleic acids, and refers to an isolated nucleicacid molecule or construct, e.g., messenger RNA (mRNA) or plasmid DNA(pDNA). A polynucleotide can comprise a conventional phosphodiester bondor a non-conventional bond (e.g., an amide bond, such as found inpeptide nucleic acids (PNA)). The term “nucleic acid” refers to any oneor more nucleic acid segments, e.g., DNA or RNA fragments, present in apolynucleotide. By “isolated” nucleic acid or polynucleotide is intendeda nucleic acid molecule, DNA or RNA, that has been removed from itsnative environment. For example, a recombinant polynucleotide encodingan anti-ILT7 binding molecule, e.g., an antibody or antigen bindingfragment thereof, contained in a vector is considered isolated for thepurposes of the present invention. Further examples of an isolatedpolynucleotide include recombinant polynucleotides maintained inheterologous host cells or purified (partially or substantially)polynucleotides in solution. Isolated RNA molecules include in vivo orin vitro RNA transcripts of polynucleotides of the present invention.Isolated polynucleotides or nucleic acids according to the presentinvention further include such molecules produced synthetically. Inaddition, a polynucleotide or a nucleic acid can be or can include aregulatory element such as a promoter, ribosome binding site, or atranscription terminator.

As used herein, a “coding region” is a portion of nucleic acid thatconsists of codons translated into amino acids. Although a “stop codon”(TAG, TGA, or TAA) is not translated into an amino acid, it can beconsidered to be part of a coding region, but any flanking sequences,for example promoters, ribosome binding sites, transcriptionalterminators, introns, and the like, are not part of a coding region. Twoor more coding regions of the present invention can be present in asingle polynucleotide construct, e.g., on a single vector, or inseparate polynucleotide constructs, e.g., on separate (different)vectors. Furthermore, any vector can contain a single coding region, orcan comprise two or more coding regions, e.g., a single vector canseparately encode an immunoglobulin heavy chain variable region and animmunoglobulin light chain variable region. In addition, a vector,polynucleotide, or nucleic acid of the invention can encode heterologouscoding regions, either fused or unfused to a nucleic acid encoding ananti-ILT7 antibody or fragment, variant, or derivative thereof.Heterologous coding regions include without limitation specializedelements or motifs, such as a secretory signal peptide or a heterologousfunctional domain.

In certain embodiments, the polynucleotide or nucleic acid is DNA. Inthe case of DNA, a polynucleotide comprising a nucleic acid that encodesa polypeptide normally can include a promoter and/or other transcriptionor translation control elements operably associated with one or morecoding regions. An operable association is when a coding region for agene product, e.g., a polypeptide, is associated with one or moreregulatory sequences in such a way as to place expression of the geneproduct under the influence or control of the regulatory sequence(s).Two DNA fragments (such as a polypeptide coding region and a promoterassociated therewith) are “operably associated” if induction of promoterfunction results in the transcription of mRNA encoding the desired geneproduct and if the nature of the linkage between the two DNA fragmentsdoes not interfere with the ability of the expression regulatorysequences to direct the expression of the gene product or interfere withthe ability of the DNA template to be transcribed. Thus, a promoterregion would be operably associated with a nucleic acid encoding apolypeptide if the promoter was capable of effecting transcription ofthat nucleic acid. The promoter can be a cell-specific promoter thatdirects substantial transcription of the DNA only in predeterminedcells. Other transcription control elements, besides a promoter, forexample enhancers, operators, repressors, and transcription terminationsignals, can be operably associated with the polynucleotide to directcell-specific transcription. Suitable promoters and other transcriptioncontrol regions are disclosed herein.

A variety of transcription control regions are known to those skilled inthe art. These include, without limitation, transcription controlregions that function in vertebrate cells, such as, but not limited to,promoter and enhancer segments from cytomegaloviruses (the immediateearly promoter, in conjunction with intron-A), simian virus 40 (theearly promoter), and retroviruses (such as Rous sarcoma virus). Othertranscription control regions include those derived from vertebrategenes such as actin, heat shock protein, bovine growth hormone andrabbit β-globin, as well as other sequences capable of controlling geneexpression in eukaryotic cells. Additional suitable transcriptioncontrol regions include tissue-specific promoters and enhancers as wellas lymphokine-inducible promoters (e.g., promoters inducible byinterferons or interleukins).

Similarly, a variety of translation control elements are known to thoseof ordinary skill in the art. These include, but are not limited to,ribosome binding sites, translation initiation and termination codons,and elements derived from picornaviruses (particularly an internalribosome entry site, or IRES, also referred to as a CITE sequence).

In other embodiments, a polynucleotide of the present invention is RNA,for example, in the form of messenger RNA (mRNA).

Polynucleotide and nucleic acid coding regions of the present inventioncan be associated with additional coding regions that encode secretoryor signal peptides, which direct the secretion of a polypeptide encodedby a polynucleotide of the present invention. According to the signalhypothesis, proteins secreted by mammalian cells have a signal peptideor secretory leader sequence that is cleaved from the mature proteinonce export of the growing protein chain across the rough endoplasmicreticulum has been initiated. Those of ordinary skill in the art areaware that polypeptides secreted by vertebrate cells generally have asignal peptide fused to the N-terminus of the polypeptide, which iscleaved from the complete or “full length” polypeptide to produce asecreted or “mature” form of the polypeptide. In certain embodiments,the native signal peptide, e.g., an immunoglobulin heavy chain or lightchain signal peptide is used, or a functional derivative of thatsequence that retains the ability to direct the secretion of thepolypeptide that is operably associated with it. Alternatively, aheterologous mammalian signal peptide, or a functional derivativethereof, can be used. For example, the wild-type leader sequence can besubstituted with the leader sequence of human tissue plasminogenactivator (TPA) or mouse β-glucuronidase.

A “binding molecule” or “antigen binding molecule” of the presentinvention refers in its broadest sense to a molecule that specificallybinds an antigenic determinant. In one embodiment, the binding moleculespecifically binds to ILT7, e.g., full length ILT7 or mature ILT7. Inanother embodiment, a binding molecule of the invention is an antibodyor an antigen-binding fragment thereof. In another embodiment, a bindingmolecule of the invention comprises at least one heavy or light chainCDR of a reference antibody molecule. In another embodiment, a bindingmolecule of the invention comprises at least two CDRs from one or morereference antibody molecules. In another embodiment, a binding moleculeof the invention comprises at least three CDRs from one or morereference antibody molecules. In another embodiment, a binding moleculeof the invention comprises at least four CDRs from one or more referenceantibody molecules. In another embodiment, a binding molecule of theinvention comprises at least five CDRs from one or more referenceantibody molecules. In another embodiment, a binding molecule of theinvention comprises at least six CDRs from one or more referenceantibody molecules. In certain embodiments, the reference antibodymolecule is 7C7, ILT70080, ILT70080.1-ILT70080.7, ILT70083,ILT70083.1-ILT70083.9, ILT70089, ILT70100, ILT70137, ILT70142, ILT70144,or ILT70052.

The present invention is directed to certain anti-ILT7 antibodies, orantigen-binding fragments, variants, or derivatives thereof. The term“antibody” means an immunoglobulin molecule that recognizes andspecifically binds to a target, such as a protein, polypeptide, peptide,carbohydrate, polynucleotide, lipid, or combinations of the foregoingthrough at least one antigen recognition site within the variable regionof the immunoglobulin molecule. As used herein, the term “antibody”encompasses intact polyclonal antibodies, intact monoclonal antibodies,chimeric antibodies, humanized antibodies, human antibodies, fusionproteins comprising an antibody, and any other modified immunoglobulinmolecule so long as the antibodies exhibit the desired biologicalactivity. An antibody can be of any the five major classes ofimmunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes)thereof (e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), based on theidentity of their heavy-chain constant domains referred to as alpha,delta, epsilon, gamma, and mu, respectively. The different classes ofimmunoglobulins have different and well known subunit structures andthree-dimensional configurations. Antibodies can be naked or conjugatedto other molecules such as toxins, radioisotopes, etc.

The term “antibody fragment” or “antibody fragment thereof” refers to aportion of an intact antibody. An “antigen-binding fragment” or“antigen-binding fragment thereof” refers to a portion of an intactantibody that binds to an antigen. An antigen-binding fragment cancontain the antigenic determining variable regions of an intactantibody. Examples of antibody fragments include, but are not limited toFab, Fab′, F(ab′)2, and Fv fragments, linear antibodies, scFvs, andsingle chain antibodies.

As used herein, “human” or “fully human” antibodies include antibodieshaving the amino acid sequence of a human immunoglobulin and includeantibodies isolated from human immunoglobulin libraries or from animalstransgenic for one or more human immunoglobulins and that do not expressendogenous immunoglobulins, as described infra and, for example, in U.S.Pat. No. 5,939,598 by Kucherlapati et al. Completely human antibodiesare particularly desirable for therapeutic treatment of human patients.

Human antibodies can be made by variety of methods known in the artincluding phage display methods using antibody libraries derived fromhuman immunoglobulin sequences as described in Vaughan et al., Nat.Biotech. 14:309-314 (1996), Sheets et al., Proc. Nat'l. Acad. Sci.95:6157-6162 (1998), Hoogenboom and Winter, J. Mol. Biol. 227:381(1992), and Marks et al., J. Mol. Biol. 222:581 (1991)). Additionalexamples of phage display methods that can be used to make and useantibodies include those disclosed in Rothe et al., J. Mol. Biol.,376:1182 (2008), Brinkman et al., J. Immunol. Methods 182:41-50 (1995);Ames et al., J. Immunol. Methods 184:177-186 (1995); Kettleborough etal., Eur. J. Immunol. 24:952-958 (1994); Persic et al., Gene 187:9-18(1997); Burton et al., Advances in Immunology 57:191-280 (1994); PCTApplication No. PCT/GB91/01134; PCT publications WO 90/02809; WO91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO95/20401; and U.S. Pat. Nos. 6,172,197; 5,885,793, 6,521,404; 6,544,731;6,555,313; 6,582,915; 6,593,081; 6,300,064; 6,653,068; 6,706,484;7,264,963; 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908;5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225;5,658,727; 5,733,743 and 5,969,108; each of which is incorporated hereinby reference in its entirety.

In addition, as known in the art, human antibodies can be produced usingtransgenic mice which are incapable of expressing functional endogenousimmunoglobulins, but which can express human immunoglobulin genes. Foran overview of this technology, see Lonberg and Huszar, Int. Rev.Immunol. 13:65-93 (1995).

Further techniques available in the art of antibody engineering havemade it possible to isolate human antibodies or fragments thereof. Forexample, human hybridomas can be made as described by Kontermann andSefan. Antibody Engineering, Springer Laboratory Manuals (2001). Fullyhuman antibodies can likewise be produced by various displaytechnologies, e.g., phage display or other viral display systems. Inphage display methods, functional antibody domains are displayed on thesurface of phage particles which carry the polynucleotide sequencesencoding them. For example, DNA sequences encoding VH and VL regions areamplified from animal cDNA libraries (e.g., human or murine cDNAlibraries of lymphoid tissues) or synthetic cDNA libraries. In certainembodiments, the DNA encoding the VH and VL regions are joined togetherby an scFv linker by PCR and cloned into a phagemid vector (e.g., pCANTAB 6 or pComb 3 HSS). The vector is electroporated in E. coli andthe E. coli is infected with helper phage. Phage used in these methodsare typically filamentous phage including fd and M13, and the VH or VLregions are usually recombinantly fused to either the phage gene III orgene VIII. Phage expressing an antigen binding domain that binds to anantigen of interest (i.e., ILT7) can be selected or identified withantigen, e.g., using labeled antigen or antigen bound or captured to asolid surface or bead.

“Human” or “fully human” antibodies also include antibodies comprisingat least the variable domain of a heavy chain, or at least the variabledomains of a heavy chain and a light chain, where the variable domain(s)have the amino acid sequence of human immunoglobulin variable domain(s).

“Human” or “fully human” antibodies also include “human” or “fullyhuman” antibodies, as described above, that comprise, consistessentially of, or consist of, variants (including derivatives) ofantibody molecules (e.g., the VH regions and/or VL regions) describedherein, which antibodies or antigen-binding fragments, variants, orderivatives thereof immunospecifically bind to a ILT7 polypeptide orfragment or variant thereof. Standard techniques known to those of skillin the art can be used to introduce mutations in the nucleotide sequenceencoding a human anti-ILT7 antibody, including, but not limited to,site-directed mutagenesis and PCR-mediated mutagenesis which result inamino acid substitutions. The variants (including derivatives) canencode less than 50 amino acid substitutions, less than 40 amino acidsubstitutions, less than 30 amino acid substitutions, less than 25 aminoacid substitutions, less than 20 amino acid substitutions, less than 15amino acid substitutions, less than 10 amino acid substitutions, lessthan 5 amino acid substitutions, less than 4 amino acid substitutions,less than 3 amino acid substitutions, or less than 2 amino acidsubstitutions relative to the reference VH region, VHCDR1, VHCDR2,VHCDR3, VL region, VLCDR1, VLCDR2, or VLCDR3.

In certain embodiments, the amino acid substitutions are conservativeamino acid substitutions, discussed further below. Alternatively,mutations can be introduced randomly along all or part of the codingsequence, such as by saturation mutagenesis, and the resultant mutantscan be screened for biological activity to identify mutants that retainactivity (e.g., the ability to bind a ILT7 polypeptide, e.g., human,primate, murine, or any combination of human, primate and murine ILT7).Such variants (or derivatives thereof) of “human” or “fully human”antibodies can also be referred to as human or fully human antibodiesthat are “optimized” or “optimized for antigen binding” and includeantibodies that have improved affinity to antigen.

Basic immunoglobulin structures in vertebrate systems are relativelywell understood. See, e.g., Harlow et al. (1988) Antibodies: ALaboratory Manual (2nd ed.; Cold Spring Harbor Laboratory Press).

As will be discussed in more detail below, the term “immunoglobulin”comprises various broad classes of polypeptides that can bedistinguished biochemically. Those skilled in the art will appreciatethat heavy chains are classified as gamma, mu, alpha, delta, or epsilon,(γ, μ, α, δ, ε) with some subclasses among them (e.g., γ1-γ4). It is thenature of this chain that determines the “class” of the antibody as IgG,IgM, IgA IgG, or IgE, respectively. The immunoglobulin subclasses(isotypes) e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc. are wellcharacterized and are known to confer functional specialization.Modified versions of each of these classes and isotypes are readilydiscernable to the skilled artisan in view of the instant disclosureand, accordingly, are within the scope of the instant invention. Whilethe following discussion will generally be directed to the IgG class ofimmunoglobulin molecules, all immunoglobulin classes are clearly withinthe scope of the present invention. With regard to IgG, a standardimmunoglobulin molecule comprises two identical light chain polypeptidesof molecular weight approximately 23,000 Daltons, and two identicalheavy chain polypeptides of molecular weight 53,000-70,000. The fourchains are typically joined by disulfide bonds in a “Y” configurationwherein the light chains bracket the heavy chains starting at the mouthof the “Y” and continuing through the variable region.

Light chains are classified as either kappa or lambda (x, X). Each heavychain class can be bound with either a kappa or lambda light chain. Ingeneral, the light and heavy chains are covalently bonded to each other,and the “tail” portions of the two heavy chains are bonded to each otherby covalent disulfide linkages or non-covalent linkages when theimmunoglobulins are generated either by hybridomas, B cells orgenetically engineered host cells. In the heavy chain, the amino acidsequences run from an N-terminus at the forked ends of the Yconfiguration to the C-terminus at the bottom of each chain.

The base of the antibody “Y” is called the Fc (Fragment, crystallizable)region, and is composed of two heavy chains that contribute two or threeconstant domains depending on the class of the antibody. Thus, the Fcregion binds to a specific class of Fc receptors, and other immunemolecules, such as complement proteins. Both the light and heavy chainsare divided into regions of structural and functional homology. Theterms “constant” and “variable” are used functionally. In this regard,it will be appreciated that the variable domains of both the light (VLor VK) and heavy (VH) chain portions determine antigen recognition andspecificity. Conversely, the constant domains of the light chain (CL)and the heavy chain (CH1, CH2 or CH3) confer important biologicalproperties such as secretion, transplacental mobility, Fc receptorbinding, complement binding, and the like. By convention the numberingof the constant region domains increases as they become more distal fromthe antigen binding site or amino-terminus of the antibody. TheN-terminal portion is a variable region and at the C-terminal portion isa constant region; the CH3 and CL domains actually comprise thecarboxy-terminus of the heavy and light chain, respectively.

As indicated above, the variable region allows the antibody toselectively recognize and specifically bind epitopes on antigens. Thatis, the VL domain and VH domain, or subset of the complementaritydetermining regions (CDRs) within these variable domains, of an antibodycombine to form the variable region that defines a three dimensionalantigen binding site. This quaternary antibody structure forms theantigen binding site present at the end of each arm of the Y. Morespecifically, the antigen binding site is defined by three CDRs on eachof the VH and VL chains. In some instances, e.g., certain immunoglobulinmolecules derived from camelid species or engineered based on camelidimmunoglobulins, a complete immunoglobulin molecule can consist of heavychains only, with no light chains. See, e.g., Hamers-Casterman et al.,Nature 363:446-448 (1993).

In naturally occurring antibodies, the six “complementarity determiningregions” or “CDRs” present in each antigen binding domain are short,non-contiguous sequences of amino acids that are specifically positionedto form the antigen binding domain as the antibody assumes its threedimensional configuration in an aqueous environment. The remainder ofthe amino acids in the antigen binding domains, referred to as“framework” regions, show less inter-molecular variability. Theframework regions largely adopt a β-sheet conformation and the CDRs formloops that connect, and in some cases form part of, the β-sheetstructure. Thus, framework regions act to form a scaffold that providesfor positioning the CDRs in correct orientation by inter-chain,non-covalent interactions. The antigen binding domain formed by thepositioned CDRs defines a surface complementary to the epitope on theimmunoreactive antigen. This complementary surface promotes thenon-covalent binding of the antibody to its cognate epitope. The aminoacids comprising the CDRs and the framework regions, respectively, canbe readily identified for any given heavy or light chain variable domainby one of ordinary skill in the art, since they have been preciselydefined (see below).

In the case where there are two or more definitions of a term that isused and/or accepted within the art, the definition of the term as usedherein is intended to include all such meanings unless explicitly statedto the contrary. A specific example is the use of the term“complementarity determining region” (“CDR”) to describe thenon-contiguous antigen combining sites found within the variable regionof both heavy and light chain polypeptides. This particular region hasbeen described by Kabat et al. (1983) U.S. Dept. of Health and HumanServices, “Sequences of Proteins of Immunological Interest” and byChothia and Lesk, J. Mol. Biol. 196:901-917 (1987), which areincorporated herein by reference, where the definitions includeoverlapping or subsets of amino acid residues when compared against eachother. Nevertheless, application of either definition to refer to a CDRof an antibody or variants thereof is intended to be within the scope ofthe term as defined and used herein. IMGT (ImMunoGeneTics) also providesa numbering system for the immunoglobulin variable regions, includingthe CDRs. See e.g., Lefranc, M. P. et al., Dev. Comp. Immunol. 27:55-77(2003), which is herein incorporated by reference. The IMGTnumbering system was based on an alignment of more than 5,000 sequences,structural data, and characterization of hypervariable loops and allowsfor easy comparison of the variable and CDR regions for all species. Theappropriate amino acid residues that encompass the CDRs as defined byeach of the above cited references are set forth below in Table 1 as acomparison. The exact residue numbers that encompass a particular CDRcan vary depending on the sequence and size of the CDR. Those skilled inthe art can routinely determine which residues comprise a particular CDRgiven the variable region amino acid sequence of the antibody.

TABLE 1 CDR Definitions¹ Kabat Chothia IMGT VH CDR1 31-35 26-32 26-35 VHCDR2 50-65 52-58 51-57 VH CDR3  95-102  95-102  93-102 VL CDR1 24-3426-32 27-32 VL CDR2 50-56 50-52 50-52 VL CDR3 89-97 91-96 89-97¹Numbering of all CDR definitions in Table 1 is according to thenumbering conventions set forth by Kabat et al. (see below).

Kabat et al. also defined a numbering system for variable domainsequences that is applicable to any antibody. One of ordinary skill inthe art can unambiguously assign this system of “Kabat numbering” to anyvariable domain sequence, without reliance on any experimental databeyond the sequence itself. As used herein, “Kabat numbering” refers tothe numbering system set forth by Kabat et al. (1983) U.S. Dept. ofHealth and Human Services, “Sequence of Proteins of ImmunologicalInterest.”

Antibodies or antigen-binding fragments, variants, or derivativesthereof of the invention include, but are not limited to, polyclonal,monoclonal, mouse, human, humanized, primatized, or chimeric antibodies,single-chain antibodies, epitope-binding fragments, e.g., Fab, Fab′ andF(ab′)₂, Fd, Fvs, single-chain Fvs (scFv), disulfide-linked Fvs (sdFv),fragments comprising either a VL or VH domain, fragments produced by aFab expression library, and anti-idiotypic (anti-Id) antibodies(including, e.g., anti-Id antibodies to anti-ILT7 antibodies disclosedherein). ScFv molecules are known in the art and are described, e.g., inU.S. Pat. No. 5,892,019. Immunoglobulin or antibody molecules of theinvention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY),class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2, etc.), or subclassof immunoglobulin molecule.

As used herein, the term “heavy chain portion” includes amino acidsequences derived from an immunoglobulin heavy chain. A polypeptidecomprising a heavy chain portion comprises at least one of: a CH1domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain,a CH2 domain, a CH3 domain, or a variant or fragment thereof. Forexample, a binding polypeptide for use in the invention can comprise apolypeptide chain comprising a CH1 domain; a polypeptide chaincomprising a CH1 domain, at least a portion of a hinge domain, and a CH2domain; a polypeptide chain comprising a CH1 domain and a CH3 domain; apolypeptide chain comprising a CH1 domain, at least a portion of a hingedomain, and a CH3 domain, or a polypeptide chain comprising a CH1domain, at least a portion of a hinge domain, a CH2 domain, and a CH3domain. In another embodiment, a polypeptide of the invention comprisesa polypeptide chain comprising a CH3 domain. Further, a bindingpolypeptide for use in the invention can lack at least a portion of aCH2 domain (e.g., all or part of a CH2 domain). As set forth above, itwill be understood by one of ordinary skill in the art that thesedomains (e.g., the heavy chain portions) can be modified such that theyvary in amino acid sequence from the naturally occurring immunoglobulinmolecule.

In certain anti-ILT7 antibodies, or antigen-binding fragments, variants,or derivatives thereof disclosed herein, the heavy chain portions of onepolypeptide chain of a multimer are identical to those on a secondpolypeptide chain of the multimer. Alternatively, heavy chainportion-containing monomers of the invention are not identical.

The heavy chain portions of a binding molecule for use in the diagnosticand treatment methods disclosed herein can be derived from differentimmunoglobulin molecules. For example, a heavy chain portion of apolypeptide can comprise a C_(H1) domain derived from an IgG1 moleculeand a hinge region derived from an IgG3 molecule. In another example, aheavy chain portion can comprise a hinge region derived, in part, froman IgG1 molecule and, in part, from an IgG3 molecule. In anotherexample, a heavy chain portion can comprise a chimeric hinge derived, inpart, from an IgG1 molecule and, in part, from an IgG4 molecule.

As used herein, the term “light chain portion” includes amino acidsequences derived from an immunoglobulin light chain, e.g., a kappa orlambda light chain. The light chain portion can comprise at least one ofa VL or CL domain.

Anti-ILT7 antibodies, or antigen-binding fragments, variants, orderivatives thereof disclosed herein can be described or specified interms of the epitope(s) or portion(s) of an antigen, e.g., a targetpolypeptide disclosed herein (e.g., full length or mature ILT7) thatthey recognize or specifically bind. The portion of a target polypeptidethat specifically interacts with the antigen binding domain of anantibody is an “epitope,” or an “antigenic determinant.” A targetpolypeptide can comprise a single epitope, but typically comprises atleast two epitopes, and can include any number of epitopes, depending onthe size, conformation, and type of antigen. Furthermore, it should benoted that an “epitope” on a target polypeptide can be or can includenon-polypeptide elements, e.g., an epitope can include a carbohydrateside chain.

The minimum size of a peptide or polypeptide epitope for an antibody isthought to be about four to five amino acids. Peptide or polypeptideepitopes can contain at least seven, at least nine, or at least about 15to about 30 amino acids. Since a CDR can recognize an antigenic peptideor polypeptide in its tertiary form, the amino acids comprising anepitope need not be contiguous, and in some cases, may not even be onthe same peptide chain. A peptide or polypeptide epitope recognized byanti-ILT7 antibodies of the present invention can contain a sequence ofat least 4, at least 5, at least 6, at least 7, at least 8, at least 9,at least 10, at least 15, at least 20, at least 25, or between about 15to about 30 contiguous or non-contiguous amino acids of ILT7.

By “specifically binds,” it is generally meant that an antibody binds toan epitope via its antigen binding domain, and that the binding entailssome complementarity between the antigen binding domain and the epitope.According to this definition, an antibody is said to “specifically bind”to an epitope when it binds to that epitope, via its antigen bindingdomain more readily than it would bind to a random, unrelated epitope.The term “specificity” is used herein to qualify the relative affinityby which a certain antibody binds to a certain epitope. For example,antibody “A” can be deemed to have a higher specificity for a givenepitope than antibody “B,” or antibody “A” can be said to bind toepitope “C” with a higher specificity than it has for related epitope“D.”

By “preferentially binds,” it is meant that the antibody specificallybinds to an epitope more readily than it would bind to a related,similar, homologous, or analogous epitope. Thus, an antibody that“preferentially binds” to a given epitope would more likely bind to thatepitope than to a related epitope, even though such an antibody cancross-react with the related epitope.

By way of non-limiting example, an antibody can be considered to bind afirst epitope preferentially if it binds said first epitope with adissociation constant (K_(D)) that is less than the antibody's K_(D) forthe second epitope. In another non-limiting example, an antibody can beconsidered to bind a first antigen preferentially if it binds the firstepitope with an affinity that is at least one order of magnitude lessthan the antibody's K_(D) for the second epitope. In anothernon-limiting example, an antibody can be considered to bind a firstepitope preferentially if it binds the first epitope with an affinitythat is at least two orders of magnitude less than the antibody's K_(D)for the second epitope.

In another non-limiting example, an antibody can be considered to bind afirst epitope preferentially if it binds the first epitope with an offrate (k(off)) that is less than the antibody's k(off) for the secondepitope. In another non-limiting example, an antibody can be consideredto bind a first epitope preferentially if it binds the first epitopewith an affinity that is at least one order of magnitude less than theantibody's k(off) for the second epitope. In another non-limitingexample, an antibody can be considered to bind a first epitopepreferentially if it binds the first epitope with an affinity that is atleast two orders of magnitude less than the antibody's k(off) for thesecond epitope. An antibody or antigen-binding fragment, variant, orderivative thereof disclosed herein can be said to bind a targetpolypeptide disclosed herein (e.g., ILT7, e.g., human, primate, murine,or any combination of human, primate and murine ILT7) or a fragment orvariant thereof with an off rate (k(off)) of less than or equal to5×10⁻² sec-1, 10⁻² sec-1, 5×10⁻³ sec⁻¹ or 10⁻³ sec⁻¹. An antibody of theinvention can be said to bind a target polypeptide disclosed herein(e.g., ILT7, e.g., human, primate, murine, or any combination of human,primate and murine ILT7) or a fragment or variant thereof with an offrate (k(off)) less than or equal to 5×10⁻⁴ sec⁻¹, 10⁻⁴ sec⁻¹, 5×10⁻⁵sec⁻¹, or 10⁻⁵ sec⁻¹, 5×10⁻⁶ sec⁻¹, 10⁻⁶ sec⁻¹, 5×10⁻⁷ sec⁻¹ or 10⁻⁷sec⁻¹.

An antibody or antigen-binding fragment, variant, or derivative thereofdisclosed herein can be said to bind a target polypeptide disclosedherein (e.g., ILT7, e.g., human, primate, murine, or any combination ofhuman, primate and murine ILT7) or a fragment or variant thereof with anon rate (k(on)) of greater than or equal to 10³ M⁻¹ sec⁻¹, 5×10³ M⁻¹sec⁻¹, 10⁴ M⁻¹ sec⁻¹ or 5×10⁴ M⁻¹ sec⁻¹. An antibody of the inventioncan bind a target polypeptide disclosed herein (e.g., ILT7, e.g., human,primate, murine, or any combination of human, primate and murine ILT7)or a fragment or variant thereof with an on rate (k(on)) greater than orequal to 10⁵ M⁻¹ sec⁻¹, 5×10⁵ M⁻¹ sec⁻¹, 10⁶ M⁻¹ sec⁻¹, or 5×10⁶ M⁻¹sec⁻¹ or 10⁷ M⁻¹ sec⁻¹.

An antibody is said to competitively inhibit binding of a referenceantibody to a given epitope if it preferentially binds to that epitopeor an overlapping epitope to the extent that it blocks, to some degree,binding of the reference antibody to the epitope. Competitive inhibitioncan be determined by any method known in the art, for example,competition ELISA assays. An antibody can be said to competitivelyinhibit binding of the reference antibody to a given epitope by at least90%, at least 80%, at least 70%, at least 60%, or at least 50%.

As used herein, the term “affinity” refers to a measure of the strengthof the binding of an individual epitope with the CDR of animmunoglobulin molecule. See, e.g., Harlow et al. (1988) Antibodies: ALaboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed.) pages27-28. As used herein, the term “avidity” refers to the overallstability of the complex between a population of immunoglobulins and anantigen, that is, the functional combining strength of an immunoglobulinmixture with the antigen. See, e.g., Harlow at pages 29-34. Avidity isrelated to both the affinity of individual immunoglobulin molecules inthe population with specific epitopes, and also the valencies of theimmunoglobulins and the antigen. For example, the interaction between abivalent monoclonal antibody and an antigen with a highly repeatingepitope structure, such as a polymer, would be one of high avidity.

Anti-ILT7 antibodies or antigen-binding fragments, variants, orderivatives thereof of the invention can also be described or specifiedin terms of their cross-reactivity. As used herein, the term“cross-reactivity” refers to the ability of an antibody, specific forone antigen, to react with a second antigen; a measure of relatednessbetween two different antigenic substances. Thus, an antibody is crossreactive if it binds to an epitope other than the one that induced itsformation. The cross reactive epitope generally contains many of thesame complementary structural features as the inducing epitope, and insome cases, can actually fit better than the original.

For example, certain antibodies have some degree of cross-reactivity, inthat they bind related, but non-identical epitopes, e.g., epitopes withat least 95%, at least 90%, at least 85%, at least 80%, at least 75%, atleast 70%, at least 65%, at least 60%, at least 55%, and at least 50%identity (as calculated using methods known in the art and describedherein) to a reference epitope. An antibody can be said to have littleor no cross-reactivity if it does not bind epitopes with less than 95%,less than 90%, less than 85%, less than 80%, less than 75%, less than70%, less than 65%, less than 60%, less than 55%, and less than 50%identity (as calculated using methods known in the art and describedherein) to a reference epitope. An antibody can be deemed “highlyspecific” for a certain epitope, if it does not bind any other analog,ortholog, or homolog of that epitope.

Anti-ILT7 binding molecules, e.g., antibodies or antigen-bindingfragments, variants or derivatives thereof of the invention can also bedescribed or specified in terms of their binding affinity to apolypeptide of the invention, e.g., ILT7, e.g., human, primate, murine,or any combination of human, primate and murine ILT7. Useful bindingaffinities include those with a dissociation constant or Kd less than5×10⁻² M, 10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M,5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁷ M, 10⁻⁷M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M,5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M,10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, or 10⁻¹⁵ M.

In some embodiments, the antibody binds to human ILT7 with adissociation constant or Kd less than 1 nM. In some embodiments, theantibody binds to cynomolgus ILT7 with a dissociation constant or Kdless than 5 nM. In some embodiments, the antibody binds to human ILT7with a dissociation constant or Kd less than 1 nM and binds tocynomolgus ILT7 with a dissociation constant or Kd less than 5 nM.

As previously indicated, the subunit structures and three dimensionalconfiguration of the constant regions of the various immunoglobulinclasses are well known. As used herein, the term “VH domain” includesthe amino terminal variable domain of an immunoglobulin heavy chain andthe term “CH1 domain” includes the first (most amino terminal) constantregion domain of an immunoglobulin heavy chain. The CH1 domain isadjacent to the VH domain and is amino terminal to the hinge region ofan immunoglobulin heavy chain molecule.

As used herein the term “CH2 domain” includes the portion of a heavychain molecule that extends, e.g., from about residue 244 to residue 360of an antibody using conventional numbering schemes (residues 244 to360, Kabat numbering system; and residues 231-340, EU numbering system;see Kabat E A et al.). The CH2 domain is unique in that it is notclosely paired with another domain. Rather, two N-linked branchedcarbohydrate chains are interposed between the two CH2 domains of anintact native IgG molecule. It is also well documented that the CH3domain extends from the CH2 domain to the C-terminal of the IgG moleculeand comprises approximately 108 residues.

As used herein, the term “hinge region” includes the portion of a heavychain molecule that joins the CH1 domain to the CH2 domain. This hingeregion comprises approximately 25 residues and is flexible, thusallowing the two N-terminal antigen binding regions to moveindependently. Hinge regions can be subdivided into three distinctdomains: upper, middle, and lower hinge domains (Roux et al., J.Immunol. 161:4083 (1998)).

As used herein the term “disulfide bond” includes the covalent bondformed between two sulfur atoms. The amino acid cysteine comprises athiol group that can form a disulfide bond or bridge with a second thiolgroup. In most naturally occurring IgG molecules, the CH1 and CL regionsare linked by a disulfide bond and the two heavy chains are linked bytwo disulfide bonds at positions corresponding to 239 and 242 using theKabat numbering system (position 226 or 229, EU numbering system).

As used herein, the term “chimeric antibody” will be held to mean anyantibody wherein the immunoreactive region or site is obtained orderived from a first species and the constant region (which can beintact, partial or modified in accordance with the instant invention) isobtained from a second species. In certain embodiments the targetbinding region or site will be from a non-human source (e.g., mouse orprimate) and the constant region is human.

As used herein, the term “engineered antibody” refers to an antibody inwhich the variable domain in either the heavy or light chain or both isaltered by at least partial replacement of one or more CDRs from anantibody of known specificity and, if necessary, by partial frameworkregion replacement and sequence changing. Although the CDRs can bederived from an antibody of the same class or even subclass as theantibody from which the framework regions are derived, it is envisagedthat the CDRs will be derived from an antibody of different class orfrom an antibody from a different species. An engineered antibody inwhich one or more “donor” CDRs from a non-human antibody of knownspecificity is grafted into a human heavy or light chain frameworkregion is referred to herein as a “humanized antibody.” It may not benecessary to replace all of the CDRs with the complete CDRs from thedonor variable domain to transfer the antigen binding capacity of onevariable domain to another. Rather, it may only be necessary to transferthose residues that are necessary to maintain the activity of the targetbinding site.

It is further recognized that the framework regions within the variabledomain in a heavy or light chain, or both, of a humanized antibody cancomprise solely residues of human origin, in which case these frameworkregions of the humanized antibody are referred to as “fully humanframework regions.” Alternatively, one or more residues of the frameworkregion(s) of the donor variable domain can be engineered within thecorresponding position of the human framework region(s) of a variabledomain in a heavy or light chain, or both, of a humanized antibody ifnecessary to maintain proper binding or to enhance binding to the ILT7antigen. A human framework region that has been engineered in thismanner would thus comprise a mixture of human and donor frameworkresidues, and is referred to herein as a “partially human frameworkregion.”

For example, humanization of an anti-ILT7 antibody can be essentiallyperformed following the method of Winter and co-workers (Jones et al.,Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988);Verhoeyen et al., Science 239:1534-1536 (1988)), by substituting rodentor mutant rodent anti-ILT7 CDRs or CDR sequences into the correspondingsequences of a human antibody. See also U.S. Pat. Nos. 5,225,539;5,585,089; 5,693,761; 5,693,762; 5,859,205; herein incorporated byreference. The resulting humanized anti-ILT7 antibody would comprise atleast one rodent or mutant rodent CDR within the fully human frameworkregions of the variable domain of the heavy and/or light chain of thehumanized antibody. In some instances, residues within the frameworkregions of one or more variable domains of the humanized anti-ILT7antibody are replaced by corresponding non-human (for example, rodent)residues (see, for example, U.S. Pat. Nos. 5,585,089; 5,693,761;5,693,762; and 6,180,370), in which case the resulting humanizedanti-ILT7 antibody would comprise partially human framework regionswithin the variable domain of the heavy and/or light chain.

Furthermore, humanized antibodies can comprise residues that are notfound in the recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance (e.g., toobtain desired affinity). In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDRs correspond tothose of a non-human immunoglobulin and all or substantially all of theframework regions are those of a human immunoglobulin sequence. Thehumanized antibody optionally also will comprise at least a portion ofan immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details see Jones et al., Nature 331:522-525(1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr.Op. Struct. Biol. 2:593-596 (1992); herein incorporated by reference.Accordingly, such “humanized” antibodies can include antibodies whereinsubstantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanized antibodies are typically human antibodies in whichsome CDR residues and possibly some framework residues are substitutedby residues from analogous sites in rodent antibodies. See, for example,U.S. Pat. Nos. 5,225,539; 5,585,089; 5,693,761; 5,693,762; 5,859,205.See also U.S. Pat. No. 6,180,370, and International Publication No. WO01/27160, where humanized antibodies and techniques for producinghumanized antibodies having improved affinity for a predeterminedantigen are disclosed.

As used herein, the terms “linked,” “fused,” or “fusion” are usedinterchangeably. These terms refer to the joining together of two moreelements or components, by whatever means including chemical conjugationor recombinant means. An “in-frame fusion” refers to the joining of twoor more polynucleotide open reading frames (ORFs) to form a continuouslonger ORF, in a manner that maintains the correct translational readingframe of the original ORFs. Thus, a recombinant fusion protein is asingle protein containing two or more segments that correspond topolypeptides encoded by the original ORFs (which segments are notnormally so joined in nature). Although the reading frame is thus madecontinuous throughout the fused segments, the segments can be physicallyor spatially separated by, for example, in-frame linker sequence. Forexample, polynucleotides encoding the CDRs of an immunoglobulin variableregion can be fused, in-frame, but be separated by a polynucleotideencoding at least one immunoglobulin framework region or additional CDRregions, as long as the “fused” CDRs are co-translated as part of acontinuous polypeptide.

In the context of polypeptides, a “linear sequence” or a “sequence” isan order of amino acids in a polypeptide in an amino to carboxylterminal direction in which residues that neighbor each other in thesequence are contiguous in the primary structure of the polypeptide.

The term “expression” as used herein refers to a process by which a geneproduces a biochemical, for example, a polypeptide. The process includesany manifestation of the functional presence of the gene within the cellincluding, without limitation, gene knockdown as well as both transientexpression and stable expression. It includes without limitationtranscription of the gene into messenger RNA (mRNA), and the translationof such mRNA into polypeptide(s). If the final desired product is abiochemical, expression includes the creation of that biochemical andany precursors. Expression of a gene produces a “gene product.” As usedherein, a gene product can be either a nucleic acid, e.g., a messengerRNA produced by transcription of a gene, or a polypeptide which istranslated from a transcript. Gene products described herein furtherinclude nucleic acids with post transcriptional modifications, e.g.,polyadenylation, or polypeptides with post translational modifications,e.g., methylation, glycosylation, the addition of lipids, associationwith other protein subunits, proteolytic cleavage, and the like.

As used herein, the terms “treat” or “treatment” refer to boththerapeutic treatment and prophylactic or preventative measures, whereinthe object is to prevent or slow down (lessen) an undesiredphysiological change or disorder, such as the progression of anautoimmune condition. Beneficial or desired clinical results include,but are not limited to, alleviation of symptoms, diminishment of extentof disease, stabilized (i.e., not worsening) state of disease, delay orslowing of disease progression, amelioration or palliation of thedisease state, and remission (whether partial or total), whetherdetectable or undetectable. “Treatment” can also mean prolongingsurvival as compared to expected survival if not receiving treatment.Those in need of treatment include those already with the condition ordisorder as well as those prone to have the condition or disorder orthose in which the condition or disorder is to be prevented.

By “subject” or “individual” or “animal” or “patient” or “mammal,” ismeant any subject, particularly a mammalian subject, for whom diagnosis,prognosis, or therapy is desired. Mammalian subjects include humans,domestic animals, farm animals, and zoo, sports, or pet animals such asdogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, andso on.

As used herein, phrases such as “a subject that would benefit fromadministration of an anti-ILT7 antibody” and “an animal in need oftreatment” includes subjects, such as mammalian subjects, that wouldbenefit from administration of an anti-ILT7 antibody used, e.g., fordetection of an anti-ILT7 polypeptide (e.g., for a diagnostic procedure)and/or from treatment, i.e., palliation or prevention of a disease, withan anti-ILT7 antibody.

II. ILT7

As used herein, the terms “ILT7” and “ILT7 polypeptide” are usedinterchangably. In certain embodiments, ILT7 is full length. In anotherembodiment, ILT7 is mature ILT7 (amino acids 24-499). In otherembodiments, ILT7 can include a full length ILT7, a fragment thereof, ora ILT7 variant polypeptide, wherein the fragment of ILT7 or ILT7 variantpolypeptide retains some or all functional properties of active ILT7.

Full length human ILT7 is a 499 amino acid protein (Accession No.P59901), containing a signal peptide (amino acids 1-23), anextracellular domain (amino acids 24-446), a transmembrane domain (aminoacids 447-467), and a cytoplasmic domain (amino acids 468-499). Theextracellular domain includes four immunoglobulin-like C2 domains (aminoacids 24-118, 123-213, 224-313, and 324-413). ILT7 is a member of theimmunoglobulin-like transcript (ILT) or leukocyte immunoglobulin-likereceptor (LIR) gene family. The sequence of cynomolgus ILT7 is providedas SEQ ID NO:292:

(SEQ ID NO: 292) PRTHMQAENLLKPILWAEPGPVIIWKKPVTIWCQGTLEAQEYRLDKEGNSISRHMLKTLESENKAKFSIPSMMWEHAGRYHCYYQSPAGWSEPSDPLELVVTAYSRPSLSALPSPVVTSGVNVTLRCASRLGLGRFTLIEEGDHRLSWTLDSHQHNHGKFQALFPVGPLTFSNRGTFRCYGYENNTPYVWSEPSDPLQLLVSGVSRKPSLLTLQGPVVAPGDNLTLQCGSDVGYIRYALYKEGGDGLPQRPGQQSQAGLSQASFTLNPVRGSHGGQYRCYGAHNVSSKWSAPSDPLDILIAGQIPDRPSLSVQLGPTVASGEKVTLLCQSWGPMFTFLLAKEGAAHPPLRLRSTYRAQQYQAEFPMSPVTSAHAGTYRCYGSRSSDPYLLSHSSEPLELVVSEATETLNPAQNKSDSKTAPHLQDYTVENLIRMGIAGLVLVFLGILLFEAQQSQRSPTRCSQEVNSREDNAPFRVVEPWEQI.

ILT7 is selectively expressed on a subset of peripheral bloodmononuclear cells (PBMCs) called plasmacytoid dendritic cells (pDCs).pDCs are the main source of the immunomodulatory molecule interferon(IFN)-alpha, and ILT7 plays a role in regulating the release ofIFN-alpha from these cells.

III. Anti-ILT7 Binding Molecules

In certain embodiments, the ILT-7 binding molecules provided herein areantibodies or antigen-binding fragments thereof that contain sequencesand/or properties of ILT7-binding antibodies provided herein. The SEQ IDNOs of sequences of ILT7 antibodies are provided in Table 2.

TABLE 2 ILT7 Antibody Sequence SEQ ID NOs VH VH VH VH VH VL VL VL VL VLAntibody PN PP CDR1 CDR2 CDR3 PN PP CDR1 CDR2 CDR2 SBI28 1 2 3 4 5 6 7 89 10 7C7 11 12 13 14 15 16 17 18 19 20 ILT70080 21 22 23 24 25 26 27 2829 30 ILT70080.1 31 32 33 34 35 36 37 38 39 40 ILT70080.2 41 42 43 44 4546 47 48 49 50 ILT70080.3 51 52 53 54 55 56 57 58 59 60 ILT70080.4 61 6263 64 65 66 67 68 69 70 ILT70080.5 71 72 73 74 75 76 77 78 79 80ILT70080.6 81 82 83 84 85 86 87 88 89 90 ILT70080.7 91 92 93 94 95 96 9798 99 100 ILT70083 101 102 103 104 105 106 107 108 109 110 ILT70083.1111 112 113 114 115 116 117 118 119 120 ILT70083.2 121 122 123 124 125126 127 128 129 130 ILT70083.3 131 132 133 134 135 136 137 138 139 140ILT70083.4 141 142 143 144 145 146 147 148 149 150 ILT70083.5 151 152153 154 155 156 157 158 159 160 ILT70083.6 161 162 163 164 165 166 167168 169 170 ILT70083.7 171 172 173 174 175 176 177 178 179 180ILT70083.8 181 182 183 184 185 186 187 188 189 190 ILT70083.9 191 192193 194 195 196 197 198 199 200 ILT70137 201 202 203 204 205 206 207 208209 210 ILT70052 211 212 213 214 215 216 217 218 219 220 ILT70100 221222 223 224 225 226 227 228 229 230 ILT70142 231 232 233 234 235 236 237238 239 240 ILT70144 241 242 243 244 245 246 247 248 249 250 ILT70019251 252 253 254 255 256 257 258 259 260 ILT70028 261 262 263 264 265 266267 268 269 270 ILT70076 271 272 273 274 275 276 277 278 279 280ILT70089 281 282 283 284 285 286 287 288 289 290

In certain embodiments, the binding molecules, e.g., antibodies orantigen-binding fragments, variants, or derivatives thereof, of theinvention, e.g., antibodies 7C7, ILT70080, ILT70080.1-ILT70080.7,ILT70083, ILT70083.1-ILT70083.9, ILT70089, ILT70100, ILT70137, ILT70142,ILT70144, and ILT70052, bind to ILT7 and inhibit IFN-alpha release fromplasmacytoid dendritic cells.

In certain embodiments, the antibodies of the invention compriseanti-ILT7 antibodies or antigen-binding fragments, variants, orderivatives thereof that bind to ILT7, e.g., antibodies 7C7, ILT70080,ILT70080.1-ILT70080.7, ILT70083, ILT70083.1-ILT70083.9, ILT70089,ILT70100, ILT70137, ILT70142, ILT70144, and ILT70052. In certainembodiments the anti-ILT7 antibodies bind human, primate, murine, or anycombination of human, primate and murine ILT7.

In one embodiment, the present invention provides an isolated bindingmolecule, e.g., an antibody or antigen-binding fragment, variant, orderivative thereof, which specifically binds to the same ILT7 epitope asantibody 7C7, ILT70080, ILT70080.1-ILT70080.7, ILT70083,ILT70083.1-ILT70083.9, ILT70089, ILT70100, ILT70137, ILT70142, ILT70144,or ILT70052. In another embodiment, the present invention provides anisolated binding molecule, e.g., an antibody or antigen-bindingfragment, variant, or derivative thereof, which specifically binds tothe same ILT7 epitope as an antibody comprising the VH and VL of 7C7,ILT70080, ILT70080.1-ILT70080.7, ILT70083, ILT70083.1-ILT70083.9,ILT70089, ILT70100, ILT70137, ILT70142, ILT70144, or ILT70052. Inanother embodiment, the present invention provides an isolated bindingmolecule, e.g., an antibody or antigen-binding fragment, variant, orderivative thereof, which specifically binds to the same ILT7 epitope asan antibody comprising the VH or VL of 7C7, ILT70080,ILT70080.1-ILT70080.7, ILT70083, ILT70083.1-ILT70083.9, ILT70089,ILT70100, ILT70137, ILT70142, ILT70144, or ILT70052.

In another embodiment, the present invention provides an isolatedbinding molecule, e.g., an antibody or antigen-binding fragment,variant, or derivative thereof, which specifically binds to ILT7, andcompetitively inhibits antibody 7C7, ILT70080, ILT70080.1-ILT70080.7,ILT70083, ILT70083.1-ILT70083.9, ILT70089, ILT70100, ILT70137, ILT70142,ILT70144, or ILT70052 from specifically binding to ILT7, e.g., human,primate, murine, or any combination of human, primate, and murine ILT7.In another embodiment, the present invention provides an isolatedbinding molecule, e.g., an antibody or antigen-binding fragment,variant, or derivative thereof, which specifically binds to ILT7, andcompetitively inhibits an antibody comprising the VH and VL of 7C7,ILT70080, ILT70080.1-ILT70080.7, ILT70083, ILT70083.1-ILT70083.9,ILT70089, ILT70100, ILT70137, ILT70142, ILT70144, or ILT70052 fromspecifically binding to ILT7, e.g., human, primate, murine, or anycombination of human, primate, and murine ILT7. In another embodiment,the present invention provides an isolated binding molecule, e.g., anantibody or antigen-binding fragment, variant, or derivative thereof,which specifically binds to ILT7, and competitively inhibits an antibodycomprising the VH or VL of 7C7, ILT70080, ILT70080.1-ILT70080.7,ILT70083, ILT70083.1-ILT70083.9, ILT70089, ILT70100, ILT70137, ILT70142,ILT70144, or ILT70052 from specifically binding to ILT7, e.g., human,primate, murine, or any combination of human, primate, and murine ILT7.

In certain embodiments, the binding molecule of the invention has anamino acid sequence that has at least 80%, 85%, 88%, 89%, 90%, 91%, 92%,93%, 94%, or 95% sequence identity to the amino acid sequence for thereference anti-ILT7 antibody molecule. In a further embodiment, thebinding molecule shares at least 96%, 97%, 98%, 99%, or 100% sequenceidentity to the reference antibody. In certain embodiments, thereference antibody is 7C7, ILT70080, ILT70080.1-ILT70080.7, ILT70083,ILT70083.1-ILT70083.9, ILT70089, ILT70100, ILT70137, ILT70142, ILT70144,or ILT70052.

In another embodiment, the present invention provides an isolatedantibody or antigen-binding fragment, variant, or derivative thereofcomprising, consisting essentially of, or consisting of a VH domain thathas an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a VH amino acidsequence of SEQ ID NOs: 22, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132,142, 152, 162, 172, 182, 192, 202, 212, 222, 232, 242, 252, and 262,wherein the antibody or antigen-binding fragment, variant, or derivativethereof comprising the VH domain specifically or preferentially binds toILT7. In a further embodiment, and the antibody or antigen-bindingfragment variant or derivative thereof inhibits IFN-alpha release fromplasmacytoid dendritic cells.

In a further embodiment, the present invention includes an isolatedantibody or antigen-binding fragment, variant, or derivative thereofcomprising, consisting essentially of, or consisting of a VL domain thathas an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a VL amino acidsequence of SEQ ID NOs: 27, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137,147, 157, 167, 177, 187, 197, 207, 217, 227, 237, 247, 257, or 267,wherein the antibody or antigen-binding fragment, variant, or derivativethereof comprising the VL domain specifically or preferentially binds toILT7. In a further embodiment, and the antibody or antigen-bindingfragment, variant, or derivative thereof inhibits IFN-alpha release fromplasmacytoid dendritic cells.

In a further embodiment, the present invention includes an isolatedantibody or antigen-binding fragment, variant, or derivative thereofcomprising, consisting essentially of, or consisting of a VH domain anda VL domain that have amino acid sequences that are at least 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical tothe VH and VL sequences of SEQ ID NOs: 22 and 27; 42 and 47; 52 and 57;62 and 67; 72 and 77; 82 and 87; 92 and 97; 102 and 107; 112 and 117;122 and 127; 132 and 137; 142 and 147; 152 and 157; 162 and 167; 172 and177; 182 and 187; 192 and 197; 202 and 207; 212 and 217; 222 and 227;232 and 237; 242 and 247; 252 and 257; or 262 and 267; respectivelywherein the antibody or antigen-binding fragment, variant, or derivativethereof comprising the VH and VL domains specifically or preferentiallybinds to ILT7. In a further embodiment, and the antibody orantigen-binding fragment, variant, or derivative thereof inhibitsIFN-alpha release from plasmacytoid dendritic cells.

In a further embodiment, the present invention includes an isolatedantibody or antigen-binding fragment, variant, or derivative thereofcomprising, consisting essentially of, or consisting of a VH domain anda VL domain that have the VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2,and VL-CDR3 sequences of SEQ ID NOs: 23, 24, 25, 28, 29, and 30; 43, 44,45, 48, 49, and 50; 53, 54, 55, 58, 59, and 60; 63, 64, 65, 68, 69, and70; 73, 74, 74, 78, 79, and 80; 83, 84, 85, 88, 89, and 90; 93, 94, 95,98, 99, and 100; 103, 104, 105, 108, 109, and 110; 113, 114, 115, 118,119, and 120; 123, 124, 125, 128, 129, and 130; 133, 134, 135, 138, 139,and 140; 143, 144, 145, 148, 149, and 150; 153, 154, 155, 158, 159, and160; 163, 164, 165, 168, 169, and 170; 173, 174, 175, 178, 179, and 180;183, 184, 185, 188, 189, and 190; 193, 194, 195, 198, 199, and 200; 203,204, 205, 208, 209, and 210; 213, 214, 215, 218, 219, and 220; 223, 224,225, 228, 229, and 230; 233, 234, 235, 238, 239, and 240; 243, 244, 245,248, 249, and 250; 253, 254, 255, 258, 259, and 260; 263, 264, 265, 268,269, and 270; respectively, wherein the antibody or antigen-bindingfragment, variant, or derivative thereof comprising the VH and VLdomains specifically or preferentially binds to ILT7. In a furtherembodiment, and the antibody or antigen-binding fragment, variant, orderivative thereof inhibits IFN-alpha release from plasmacytoiddendritic cells.

Suitable biologically active variants of the anti-ILT7 antibodies of theinvention can be used in the methods of the present invention. Suchvariants will retain the desired binding properties of the parentanti-ILT7 antibody. Methods for making antibody variants are generallyavailable in the art.

Methods for mutagenesis and nucleotide sequence alterations are wellknown in the art. See, for example, Walker and Gaastra, eds. (1983)Techniques in Molecular Biology (MacMillan Publishing Company, NewYork); Kunkel, Proc. Natl. Acad Sci. USA 82:488-492 (1985); Kunkel etal., Methods Enzymol. 154:367-382 (1987); Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, N.Y.); U.S.Pat. No. 4,873,192; and the references cited therein; hereinincorporated by reference. Guidance as to appropriate amino acidsubstitutions that do not affect biological activity of the polypeptideof interest can be found in the model of Dayhoff et al. (1978) in Atlasof Protein Sequence and Structure (Natl. Biomed. Res. Found.,Washington, D.C.), pp. 345-352, herein incorporated by reference in itsentirety. The model of Dayhoff et al. uses the Point Accepted Mutation(PAM) amino acid similarity matrix (PAM 250 matrix) to determinesuitable conservative amino acid substitutions. Conservativesubstitutions, such as exchanging one amino acid with another havingsimilar properties, can be beneficial. Examples of conservative aminoacid substitutions as taught by the PAM 250 matrix of the Dayhoff et al.model include, but are not limited to, Gly↔Ala, Val↔Ile↔Leu, Asp↔Glu,Lys↔Arg, Asn↔Gln, and Phe↔Trp↔Tyr.

In constructing variants of an anti-ILT7 binding molecule, e.g., anantibody or antigen-binding fragment, variant, or derivative thereof,modifications are made such that variants continue to possess thedesired properties, e.g., being capable of specifically binding to aILT7, and in certain embodiments being able to inhibit IFN-alpharelease. Obviously, any mutations made in the DNA encoding the variantpolypeptide must not place the sequence out of reading frame. In someembodiment, any mutations made in the DNA will not create complementaryregions that could produce secondary mRNA structure.

Methods for measuring the binding specificity of an anti-ILT7 bindingmolecule, e.g., an antibody or antigen-binding fragment, variant, orderivative thereof, include, but are not limited to, standardcompetitive binding assays, cytotoxicity assays, IFN release assays,ELISA assays, and the like.

When discussed herein whether any particular polypeptide, including theconstant regions, CDRs, VH domains, or VL domains disclosed herein, isat least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or even 100% identical to another polypeptide, the %identity can be determined using methods and computer programs/softwareknown in the art such as, but not limited to, the BESTFIT program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, 575 Science Drive, Madison,Wis. 53711). BESTFIT uses the local homology algorithm of Smith andWaterman (1981) Adv. Appl. Math. 2:482-489, to find the best segment ofhomology between two sequences. When using BESTFIT or any other sequencealignment program to determine whether a particular sequence is, forexample, 95% identical to a reference sequence according to the presentinvention, the parameters are set, of course, such that the percentageof identity is calculated over the full length of the referencepolypeptide sequence and that gaps in homology of up to 5% of the totalnumber of amino acids in the reference sequence are allowed.

For purposes of the present invention, percent sequence identity can bedetermined using the Smith-Waterman homology search algorithm using anaffine gap search with a gap open penalty of 12 and a gap extensionpenalty of 2, BLOSUM matrix of 62. The Smith-Waterman homology searchalgorithm is taught in Smith and Waterman (1981) Adv. Appl. Math.2:482-489. A variant can, for example, differ from a reference anti-ILT7antibody (e.g., 7C7, ILT70080, ILT70080.1-ILT70080.7, ILT70083,ILT70083.1-ILT70083.9, ILT70089, ILT70100, ILT70137, ILT70142, ILT70144,or ILT70052) by as few as 1 to 15 amino acid residues, as few as 1 to 10amino acid residues, such as 6-10, as few as 5, as few as 4, 3, 2, oreven 1 amino acid residue.

The precise chemical structure of a polypeptide capable of specificallybinding ILT7 and retaining the desired activity depends on a number offactors. As ionizable amino and carboxyl groups are present in themolecule, a particular polypeptide can be obtained as an acidic or basicsalt, or in neutral form. All such preparations that retain theirbiological activity when placed in suitable environmental conditions areincluded in the definition of anti-ILT7 antibodies as used herein.Further, the primary amino acid sequence of the polypeptide can beaugmented by derivatization using sugar moieties (glycosylation) or byother supplementary molecules such as lipids, phosphate, acetyl groupsand the like. It can also be augmented by conjugation with saccharides.Certain aspects of such augmentation are accomplished throughpost-translational processing systems of the producing host; other suchmodifications can be introduced in vitro. In any event, suchmodifications are included in the definition of an anti-ILT7 antibodyused herein so long as the desired properties of the anti-ILT7 antibodyare not destroyed. It is expected that such modifications canquantitatively or qualitatively affect the activity, either by enhancingor diminishing the activity of the polypeptide, in the various assays.Further, individual amino acid residues in the chain can be modified byoxidation, reduction, or other derivatization, and the polypeptide canbe cleaved to obtain fragments that retain activity. Such alterationsthat do not destroy the desired properties (e.g., binding specificityfor ILT7, binding affinity, and associated activity, e.g., ability toinhibit the ILT7-driven cytokine release from mast cells, endothelialcells and proliferation of TF-1 cells) do not remove the polypeptidesequence from the definition of anti-ILT7 antibodies of interest as usedherein.

The art provides substantial guidance regarding the preparation and useof polypeptide variants. In preparing variants of an anti-ILT7 bindingmolecule, e.g., an antibody or antigen-binding fragment, variant, orderivative thereof, one of skill in the art can readily determine whichmodifications to the native protein's nucleotide or amino acid sequencewill result in a variant that is suitable for use as a therapeuticallyactive component of a pharmaceutical composition used in the methods ofthe present invention.

The constant region of an anti-ILT7 antibody can be mutated to altereffector function in a number of ways. For example, see U.S. Pat. No.6,737,056B1 and U.S. Patent Application Publication No. 2004/0132101A1,which disclose Fc mutations that optimize antibody binding to Fcreceptors.

In certain anti-ILT7 antibodies, the Fc portion can be mutated todecrease effector function using techniques known in the art. Forexample, the deletion or inactivation (through point mutations or othermeans) of a constant region domain can reduce Fc receptor binding of thecirculating modified antibody. In other cases it can be that constantregion modifications consistent with the instant invention moderatecomplement binding and thus reduce the serum half life and nonspecificassociation of a conjugated cytotoxin. Yet other modifications of theconstant region can be used to modify disulfide linkages oroligosaccharide moieties that allow for enhanced localization due toincreased antigen specificity or antibody flexibility. The resultingphysiological profile, bioavailability and other biochemical effects ofthe modifications, such as biodistribution and serum half-life, caneasily be measured and quantified using well known immunologicaltechniques without undue experimentation.

Certain ILT7 antibodies provided herein are afucosylated. Antibodieslacking core fucose residues from the Fc N-glycans exhibit strong ADCCat lower concentrations with much higher efficacy compared tofucosylated counterparts, and they can evade the inhibitory effect ofserum immunoglobulin G (IgG) on ADCC through its high binding to gammareceptor IIIa (Fc FcγRIIIa).

Anti-ILT7 antibodies of the invention also include derivatives that aremodified, e.g., by the covalent attachment of any type of molecule tothe antibody such that covalent attachment does not prevent the antibodyfrom specifically binding to its cognate epitope. For example, but notby way of limitation, the antibody derivatives include antibodies thathave been modified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications can be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, etc. Additionally, the derivativecan contain one or more non-classical amino acids.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a side chain witha similar charge. Families of amino acid residues having side chainswith similar charges have been defined in the art. These familiesinclude amino acids with basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g.,alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Alternatively, mutations can beintroduced randomly along all or part of the coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forbiological activity to identify mutants that retain activity (e.g., theability to bind an anti-ILT7 polypeptide).

For example, it is possible to introduce mutations only in frameworkregions or only in CDR regions of an antibody molecule. Introducedmutations can be silent or neutral missense mutations, i.e., have no, orlittle, effect on an antibody's ability to bind antigen. These types ofmutations can be useful to optimize codon usage, or improve ahybridoma's antibody production. Alternatively, non-neutral missensemutations can alter an antibody's ability to bind antigen. The locationof most silent and neutral missense mutations is likely to be in theframework regions, while the location of most non-neutral missensemutations is likely to be in CDR, though this is not an absoluterequirement. One of skill in the art would be able to design and testmutant molecules with desired properties such as no alteration inantigen binding activity or alteration in binding activity (e.g.,improvements in antigen binding activity or change in antibodyspecificity). Following mutagenesis, the encoded protein can routinelybe expressed and the functional and/or biological activity of theencoded protein, (e.g., ability to immunospecifically bind at least oneepitope of a ILT7 polypeptide) can be determined using techniquesdescribed herein or by routinely modifying techniques known in the art.

In certain embodiments, the anti-ILT7 antibodies of the inventioncomprise at least one optimized complementarity-determining region(CDR). By “optimized CDR” is intended that the CDR has been modified andoptimized sequences selected based on the sustained or improved bindingaffinity and/or anti-ILT7 activity that is imparted to an anti-ILT7antibody comprising the optimized CDR. “Anti-ILT7 activity” can include,e.g., activity which modulates one or more of the following activitiesassociated with ILT7, e.g., ILT7-driven interferon release fromplasmacytoid dendritic cells, cytotoxicity to ILT7-expressing cells, orany other activity association with ILT7. Anti-ILT7 activity can also beattributed to a decrease in incidence or severity of diseases associatedwith ILT7 expression, including, but not limited to, certain types ofautoimmune conditions, e.g., systemic lupus erythematosus, chronicrheumatism, and psoriasis. The modifications can involve replacement ofamino acid residues within the CDR such that an anti-ILT7 antibodyretains specificity for the ILT7 antigen and has improved bindingaffinity and/or improved anti-ILT7 activity.

IV. Polynucleotides Encoding Anti-ILT7 Antibodies

The present invention also provides for nucleic acid molecules encodinganti-ILT7 antibodies of the invention, or antigen-binding fragments,variants, or derivatives thereof.

In a further embodiment, the present invention includes an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding a VH domain that has an amino acid sequence thatis at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100% identical to a reference VH domain polypeptide sequencecomprising SEQ ID NOs: 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112,122, 132, 142, 152, 162, 172, 182, 192, 202, 212, 222, 232, or 242,wherein the anti-ILT7 antibody comprising the encoded VH domainspecifically or preferentially binds to ILT7. In certain embodiments,the polynucleotide encodes an antibody or antigen-binding fragment,variant, or derivative thereof that inhibits IFN-alpha release fromplasmacytoid dendritic cells.

In a further embodiment, the present invention includes an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding a VL domain that has an amino acid sequence thatis at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100% identical to a reference VL domain polypeptide sequencecomprising SEQ ID NOs: 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117,127, 137, 147, 157, 167, 177, 187, 197, 207, 217, 227, 237, or 247,wherein the anti-ILT7 antibody comprising the encoded VL domainspecifically or preferentially binds to ILT7. In certain embodiments,the polynucleotide encodes an antibody or antigen-binding fragment,variant, or derivative thereof that inhibits IFN-alpha release fromplasmacytoid dendritic cells.

Any of the polynucleotides described above can further includeadditional nucleic acids, encoding, e.g., a signal peptide to directsecretion of the encoded polypeptide, antibody constant regions asdescribed herein, or other heterologous polypeptides as describedherein. Also, as described in more detail elsewhere herein, the presentinvention includes compositions comprising one or more of thepolynucleotides described above.

In one embodiment, the invention includes compositions comprising afirst polynucleotide and second polynucleotide wherein said firstpolynucleotide encodes a VH domain as described herein and wherein saidsecond polynucleotide encodes a VL domain as described herein.Specifically a composition can comprise, consist essentially of, orconsist of a VH domain-encoding polynucleotide, as set forth in SEQ IDNO: 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151,161, 171, 181, 191, 201, 211, 221, 231, or 241 and a VL domain-encodingpolynucleotide as set forth in SEQ ID NO: 16, 26, 36, 46, 56, 66, 76,86, 96, 106, 116, 126, 136, 146, 156, 166, 176, 186, 196, 206, 216, 226,236, or 246. A composition can also comprise, consist essentially of, orconsist of a VH domain-encoding polynucleotide that encodes the sequenceset forth in SEQ ID NO: 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112,122, 132, 142, 152, 162, 172, 182, 192, 202, 212, 222, 232, or 242, anda VL domain-encoding polynucleotide that encodes the sequence set forthin SEQ ID NO: 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137,147, 157, 167, 177, 187, 197, 207, 217, 227, 237, 247, 257, or 267. Insome embodiments, the VH domain-encoding polypeptide and the VLdomain-encoding polypeptide are on the same vector. In some embodiments,the VH domain-encoding polypeptide and the VL domain-encodingpolypeptide are on different vectors.

The present invention also includes fragments of the polynucleotides ofthe invention, as described elsewhere. Additionally polynucleotides thatencode fusion polypeptides, Fab fragments, and other derivatives, asdescribed herein, are also contemplated by the invention.

The polynucleotides can be produced or manufactured by any method knownin the art. For example, if the nucleotide sequence of the antibody isknown, a polynucleotide encoding the antibody can be assembled fromchemically synthesized oligonucleotides (e.g., as described in Kutmeieret al., Bio Techniques 17:242 (1994)), which, briefly, involves thesynthesis of overlapping oligonucleotides containing portions of thesequence encoding the antibody, annealing and ligating of thoseoligonucleotides, and then amplification of the ligated oligonucleotidesby PCR.

Alternatively, a polynucleotide encoding an anti-ILT7 antibody, orantigen-binding fragment, variant, or derivative thereof of theinvention, can be generated from nucleic acid from a suitable source. Ifa clone containing a nucleic acid encoding a particular antibody is notavailable, but the sequence of the antibody molecule is known, a nucleicacid encoding the antibody can be chemically synthesized or obtainedfrom a suitable source (e.g., an antibody cDNA library, or a cDNAlibrary generated from, or nucleic acid, e.g., poly A+RNA, isolatedfrom, any tissue or cells expressing the antibody or other anti-ILT7antibody, such as hybridoma cells selected to express an antibody) byPCR amplification using synthetic primers hybridizable to the 3′ and 5′ends of the sequence or by cloning using an oligonucleotide probespecific for the particular gene sequence to identify, e.g., a cDNAclone from a cDNA library that encodes the antibody or other anti-ILT7antibody. Amplified nucleic acids generated by PCR can then be clonedinto replicable cloning vectors using any method well known in the art.

Once the nucleotide sequence and corresponding amino acid sequence ofthe anti-ILT7 antibody, or antigen-binding fragment, variant, orderivative thereof is determined, its nucleotide sequence can bemanipulated using methods well known in the art for the manipulation ofnucleotide sequences, e.g., recombinant DNA techniques, site directedmutagenesis, PCR, etc. (see, for example, the techniques described inSambrook et al. (1990) Molecular Cloning, A Laboratory Manual (2nd ed.;Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) and Ausubel etal., eds. (1998) Current Protocols in Molecular Biology (John Wiley &Sons, NY), which are both incorporated by reference herein in theirentireties), to generate antibodies having a different amino acidsequence, for example to create amino acid substitutions, deletions,and/or insertions.

A polynucleotide encoding an anti-ILT7 binding molecule, e.g., anantibody, or antigen-binding fragment, variant, or derivative thereof,can be composed of any polyribonucleotide or polydeoxyribonucleotide,which can be unmodified RNA or DNA or modified RNA or DNA. For example,a polynucleotide encoding anti-ILT7 antibody, or antigen-bindingfragment, variant, or derivative thereof can be composed of single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that can be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, a polynucleotide encoding an anti-ILT7 binding molecule, e.g.,an antibody, or antigen-binding fragment, variant, or derivative thereofcan be composed of triple-stranded regions comprising RNA or DNA or bothRNA and DNA. A polynucleotide encoding an anti-ILT7 binding molecule,e.g., antibody, or antigen-binding fragment, variant, or derivativethereof, can also contain one or more modified bases or DNA or RNAbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications can be made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically, or metabolicallymodified forms.

An isolated polynucleotide encoding a non-natural variant of apolypeptide derived from an immunoglobulin (e.g., an immunoglobulinheavy chain portion or light chain portion) can be created byintroducing one or more nucleotide substitutions, additions or deletionsinto the nucleotide sequence of the immunoglobulin such that one or moreamino acid substitutions, additions or deletions are introduced into theencoded protein. Mutations can be introduced by standard techniques,such as site-directed mutagenesis and PCR-mediated mutagenesis.Conservative amino acid substitutions can be made at one or morenon-essential amino acid residues.

V. Fusion Proteins and Antibody Conjugates

As discussed in more detail elsewhere herein, anti-ILT7 bindingmolecules, e.g., antibodies of the invention, or antigen-bindingfragments, variants, or derivatives thereof, can further berecombinantly fused to a heterologous polypeptide at the N- orC-terminus or chemically conjugated (including covalent and non-covalentconjugations) to polypeptides or other compositions. For example,anti-ILT7 antibodies can be recombinantly fused or conjugated tomolecules useful as labels in detection assays and effector moleculessuch as heterologous polypeptides, drugs, radionuclides, or toxins. See,e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat.No. 5,314,995; and EP 396,387.

Anti-ILT7 antibodies of the invention, or antigen-binding fragments,variants, or derivatives thereof, can include derivatives that aremodified, i.e., by the covalent attachment of any type of molecule tothe antibody such that covalent attachment does not prevent the antibodyfrom binding to ILT7. For example, but not by way of limitation, theantibody derivatives include antibodies that have been modified, e.g.,by glycosylation, acetylation, pegylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, linkage to a cellular ligand or other protein, etc. Any ofnumerous chemical modifications can be carried out by known techniques,including, but not limited to specific chemical cleavage, acetylation,formylation, etc. Additionally, the derivative can contain one or morenon-classical amino acids.

Anti-ILT7 binding molecules, e.g., antibodies of the invention, orantigen-binding fragments, variants, or derivatives thereof, can becomposed of amino acids joined to each other by peptide bonds ormodified peptide bonds, i.e., peptide isosteres, and can contain aminoacids other than the 20 gene-encoded amino acids. For example, anti-ILT7antibodies can be modified by natural processes, such asposttranslational processing, or by chemical modification techniquesthat are well known in the art. Such modifications are well described inbasic texts and in more detailed monographs, as well as in a voluminousresearch literature. Modifications can occur anywhere in the anti-ILT7binding molecule, including the peptide backbone, the amino acidside-chains and the amino or carboxyl termini, or on moieties such ascarbohydrates. It will be appreciated that the same type of modificationcan be present in the same or varying degrees at several sites in agiven anti-ILT7 binding molecule. Also, a given anti-ILT7 bindingmolecule can contain many types of modifications. Anti-ILT7 bindingmolecules can be branched, for example, as a result of ubiquitination,and they can be cyclic, with or without branching. Cyclic, branched, andbranched cyclic anti-ILT7 binding molecule can result fromposttranslational natural processes or can be made by synthetic methods.Modifications include acetylation, acylation, ADP-ribosylation,amidation, covalent attachment of flavin, covalent attachment of a hememoiety, covalent attachment of a nucleotide or nucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, pegylation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination. (See, forinstance, Proteins—Structure and Molecular Properties, T. E. Creighton,W. H. Freeman and Company, NY; 2nd ed. (1993); Johnson, ed. (1983)Posttranslational Covalent Modification of Proteins (Academic Press,NY), pgs. 1-12; Seifter et al., Meth. Enzymol. 182:626-646 (1990);Rattan et al., Ann. NY Acad. Sci. 663:48-62 (1992)).

The present invention also provides for fusion proteins comprising ananti-ILT7 antibody, or antigen-binding fragment, variant, or derivativethereof, and a heterologous polypeptide. The heterologous polypeptide towhich the antibody is fused can be useful for function or is useful totarget the anti-ILT7 polypeptide expressing cells.

In one embodiment, a fusion protein of the invention comprises, consistsessentially of, or consists of, a polypeptide having the amino acidsequence of any one or more of the VH domains of an antibody of theinvention or the amino acid sequence of any one or more of the VLdomains of an antibody of the invention or fragments, variants, orderivatives thereof, and a heterologous polypeptide sequence.

In another embodiment, a fusion protein for use in the diagnostic andtreatment methods disclosed herein comprises, consists essentially of,or consists of a polypeptide having the amino acid sequence of any one,two, three of the CDRs of the VH domain of an anti-ILT7 antibody, orfragments, variants, or derivatives thereof, or the amino acid sequenceof any one, two, three of the CDRs of the VL domain an anti-ILT7antibody, or fragments, variants, or derivatives thereof, and aheterologous polypeptide sequence. In one embodiment, a fusion proteincomprises a polypeptide having the amino acid sequence of at least oneVH domain of an anti-ILT7 antibody of the invention and the amino acidsequence of at least one VL domain of an anti-ILT7 antibody of theinvention or fragments, derivatives or variants thereof, and aheterologous polypeptide sequence. In some embodiments, the VH and VLdomains of the fusion protein correspond to a single source antibody (orscFv or Fab fragment) that specifically binds at least one epitope ofILT7. In yet another embodiment, a fusion protein for use in thediagnostic and treatment methods disclosed herein comprises apolypeptide having the amino acid sequence of any one, two, three ormore of the CDRs of the VH domain of an anti-ILT7 antibody and the aminoacid sequence of any one, two, three or more of the CDRs of the VLdomain of an anti-ILT7 antibody, or fragments or variants thereof, and aheterologous polypeptide sequence. In some embodiments, two, three,four, five, six, or more of the CDR(s) of the VH domain or VL domaincorrespond to single source antibody (or scFv or Fab fragment) of theinvention. Nucleic acid molecules encoding these fusion proteins arealso encompassed by the invention.

Exemplary fusion proteins reported in the literature include fusions ofthe T cell receptor (Gascoigne et al., Proc. Natl. Acad Sci. USA84:2936-2940 (1987)); CD4 (Capon et al., Nature 337:525-531 (1989);Traunecker et al., Nature 339:68-70 (1989); Zettmeissl et al., DNA CellBiol. USA 9:347-353 (1990); and Byrn et al., Nature 344:667-670(1990));L-selectin (homing receptor) (Watson et al., J. Cell. Biol.110:2221-2229 (1990); and Watson et al., Nature 349:164-167 (1991));CD44 (Aruffo et al., Cell 61:1303-1313 (1990)); CD28 and B7 (Linsley etal., J. Exp. Med 173:721-730 (1991)); CTLA-4 (Lisley et al., J. Exp. Med174:561-569 (1991)); CD22 (Stamenkovic et al., Cell 66:1133-1144(1991)); TNF receptor (Ashkenazi et al., Proc. Natl. Acad. Sci. USA88:10535-10539 (1991); Lesslauer et al., Eur. J. Immunol. 27:2883-2886(1991); and Peppel et al., J. Exp. Med 174:1483-1489 (1991)); and IgEreceptor a (Ridgway and Gorman, J. Cell. Biol. Vol. 115, Abstract No.1448 (1991)).

As discussed elsewhere herein, anti-ILT7 binding molecules, e.g.,antibodies of the invention, or antigen-binding fragments, variants, orderivatives thereof, can be fused to heterologous polypeptides toincrease the in vivo half life of the polypeptides or for use inimmunoassays using methods known in the art. For example, in oneembodiment, PEG can be conjugated to the anti-ILT7 antibodies of theinvention to increase their half-life in vivo. See Leong et al.,Cytokine 16:106 (2001); Adv. in Drug Deliv. Rev. 54:531 (2002); or Weiret al., Biochem. Soc. Transactions 30:512 (2002).

Moreover, anti-ILT7 binding molecules, e.g., antibodies of theinvention, or antigen-binding fragments, variants, or derivativesthereof, can be fused to marker sequences, such as a peptide tofacilitate their purification or detection. In some embodiments, themarker amino acid sequence is a hexa-histidine peptide (SEQ ID NO: 299),such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 EtonAvenue, Chatsworth, Calif., 91311), among others, many of which arecommercially available. As described in Gentz et al., Proc. Natl. AcadSci. USA 86:821-824 (1989), for instance, hexa-histidine (SEQ ID NO:299) provides for convenient purification of the fusion protein. Otherpeptide tags useful for purification include, but are not limited to,the “HA” tag, which corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and the “flag”tag.

Fusion proteins can be prepared using methods that are well known in theart (see for example U.S. Pat. Nos. 5,116,964 and 5,225,538). Theprecise site at which the fusion is made can be selected empirically tooptimize the secretion or binding characteristics of the fusion protein.DNA encoding the fusion protein is then transfected into a host cell forexpression.

Anti-ILT7 binding molecules, e.g., antibodies of the present invention,or antigen-binding fragments, variants, or derivatives thereof, can beused in non-conjugated form or can be conjugated to at least one of avariety of molecules, e.g., to improve the therapeutic properties of themolecule, to facilitate target detection, or for imaging or therapy ofthe patient. Anti-ILT7 binding molecules, e.g., antibodies of theinvention, or antigen-binding fragments, variants, or derivativesthereof, can be labeled or conjugated either before or afterpurification, or when purification is performed.

In particular, anti-ILT7 antibodies of the invention, or antigen-bindingfragments, variants, or derivatives thereof, can be conjugated totherapeutic agents, prodrugs, peptides, proteins, enzymes, viruses,lipids, biological response modifiers, pharmaceutical agents, or PEG.

Those skilled in the art will appreciate that conjugates can also beassembled using a variety of techniques depending on the selected agentto be conjugated. For example, conjugates with biotin are prepared,e.g., by reacting a binding polypeptide with an activated ester ofbiotin such as the biotin N-hydroxysuccinimide ester. Similarly,conjugates with a fluorescent marker can be prepared in the presence ofa coupling agent, e.g., those listed herein, or by reaction with anisothiocyanate, such as fluorescein-isothiocyanate. Conjugates of theanti-ILT7 antibodies of the invention, or antigen-binding fragments,variants, or derivatives thereof, are prepared in an analogous manner.

The present invention further encompasses anti-ILT7 binding molecules,e.g., antibodies of the invention, or antigen-binding fragments,variants, or derivatives thereof, conjugated to a diagnostic ortherapeutic agent. The anti-ILT7 antibodies, including antigen-bindingfragments, variants, and derivatives thereof, can be used diagnosticallyto, for example, monitor the development or progression of a disease aspart of a clinical testing procedure to, e.g., determine the efficacy ofa given treatment and/or prevention regimen. For example, detection canbe facilitated by coupling the anti-ILT7 antibody, or antigen-bindingfragment, variant, or derivative thereof, to a detectable substance.Examples of detectable substances include various enzymes, prostheticgroups, fluorescent materials, luminescent materials, bioluminescentmaterials, radioactive materials, positron emitting metals using variouspositron emission tomographies, and nonradioactive paramagnetic metalions. See, for example, U.S. Pat. No. 4,741,900 for metal ions which canbe conjugated to antibodies for use as diagnostics according to thepresent invention. Examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹, ¹¹¹In, ⁹⁰Y, or ⁹⁹Tc.

An anti-ILT7 binding molecule, e.g., an antibody, or antigen-bindingfragment, variant, or derivative thereof, also can be detectably labeledby coupling it to a chemiluminescent compound. The presence of thechemiluminescent-tagged anti-ILT7 binding molecule is then determined bydetecting the presence of luminescence that arises during the course ofa chemical reaction. Examples of particularly useful chemiluminescentlabeling compounds are luminol, isoluminol, theromatic acridinium ester,imidazole, acridinium salt and oxalate ester.

One of the ways in which an anti-ILT7 antibody, or antigen-bindingfragment, variant, or derivative thereof, can be detectably labeled isby linking the same to an enzyme and using the linked product in anenzyme immunoassay (EIA) (Voller, A., “The Enzyme Linked ImmunosorbentAssay (ELISA)” Microbiological Associates Quarterly Publication,Walkersville, Md.; Diagnostic Horizons 2:1-7 (1978); Voller et al., J.Clin. Pathol. 31:507-520 (1978); Butler, Meth. Enzymol. 73:482-523(1981); Maggio, ed. (1980) Enzyme Immunoassay, CRC Press, Boca Raton,Fla.; Ishikawa et al., eds. (1981) Enzyme Immunoassay (Kgaku Shoin,Tokyo). The enzyme, which is bound to the anti-ILT7 antibody will reactwith an appropriate substrate, such as a chromogenic substrate, in sucha manner as to produce a chemical moiety which can be detected, forexample, by spectrophotometric, fluorimetric or by visual means. Enzymeswhich can be used to detectably label the antibody include, but are notlimited to, malate dehydrogenase, staphylococcal nuclease,delta-5-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. Additionally, the detection can be accomplished bycolorimetric methods which employ a chromogenic substrate for theenzyme. Detection can also be accomplished by visual comparison of theextent of enzymatic reaction of a substrate in comparison with similarlyprepared standards.

Detection may also be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling the anti-ILT7binding molecule, e.g., antibody, or antigen-binding fragment, variant,or derivative thereof, it is possible to detect the binding moleculethrough the use of a radioimmunoassay (RIA) (see, for example, Weintraub(March, 1986) Principles of Radioimmunoassays, Seventh Training Courseon Radioligand Assay Techniques (The Endocrine Society), which isincorporated by reference herein). The radioactive isotope can bedetected by means including, but not limited to, a gamma counter, ascintillation counter, or autoradiography.

An anti-ILT7 binding molecule, e.g., antibody, or antigen-bindingfragment, variant, or derivative thereof, can also be detectably labeledusing fluorescence emitting metals such as 152Eu, or others of thelanthanide series. These metals can be attached to the binding moleculeusing such metal chelating groups as diethylenetriaminepentacetic acid(DTPA) or ethylenediaminetetraacetic acid (EDTA).

Techniques for conjugating various moieties to an antibody (e.g., ananti-ILT7 antibody), or antigen-binding fragment, variant, or derivativethereof, are well known, see, e.g., Amon et al. (1985) “MonoclonalAntibodies for Immunotargeting of Drugs in Cancer Therapy,” inMonoclonal Antibodies and Cancer Therapy, ed. Reisfeld et al. (Alan R.Liss, Inc.), pp. 243-56; Hellstrom et al. (1987) “Antibodies for DrugDelivery,” in Controlled Drug Delivery, ed. Robinson et al. (2nd ed.;Marcel Dekker, Inc.), pp. 623-53); Thorpe (1985) “Antibody Carriers ofCytotoxic Agents in Cancer Therapy: A Review,” in Monoclonal Antibodies'84: Biological and Clinical Applications, ed. Pinchera et al., pp.475-506; “Analysis, Results, and Future Prospective of the TherapeuticUse of Radiolabeled Antibody in Cancer Therapy,” in MonoclonalAntibodies for Cancer Detection and Therapy, ed. Baldwin et al.,Academic Press, pp. 303-16 (1985); and Thorpe et al. (1982) “ThePreparation and Cytotoxic Properties of Antibody-Toxin Conjugates,”Immunol. Rev. 62:119-58.

VI. Expression of Antibody Polypeptides

DNA sequences that encode the light and the heavy chains of the antibodycan be made, either simultaneously or separately, using reversetranscriptase and DNA polymerase in accordance with well known methods.PCR can be initiated by consensus constant region primers or by morespecific primers based on the published heavy and light chain DNA andamino acid sequences. As discussed above, PCR also can be used toisolate DNA clones encoding the antibody light and heavy chains. In thiscase, the libraries can be screened by consensus primers or largerhomologous probes, such as mouse constant region probes.

DNA, typically plasmid DNA, can be isolated from the cells usingtechniques known in the art, restriction mapped and sequenced inaccordance with standard, well known techniques set forth in detail,e.g., in the foregoing references relating to recombinant DNAtechniques. Of course, the DNA can be synthetic according to the presentinvention at any point during the isolation process or subsequentanalysis.

Following manipulation of the isolated genetic material to provideanti-ILT7 antibodies, or antigen-binding fragments, variants, orderivatives thereof, of the invention, the polynucleotides encoding theanti-ILT7 antibodies are typically inserted in an expression vector forintroduction into host cells that can be used to produce the desiredquantity of anti-ILT7 antibody.

Recombinant expression of an antibody, or fragment, variant, orderivative thereof, e.g., a heavy or light chain of an antibody thatbinds to a target molecule described herein, e.g., ILT7, requiresconstruction of an expression vector containing a polynucleotide thatencodes the antibody. Once a polynucleotide encoding an antibodymolecule or a heavy or light chain of an antibody, or portion thereof(e.g., containing the heavy or light chain variable domain), of theinvention has been obtained, the vector for the production of theantibody molecule can be produced by recombinant DNA technology usingtechniques well known in the art. Thus, methods for preparing a proteinby expressing a polynucleotide containing an antibody encodingnucleotide sequence are described herein. Methods that are well known tothose skilled in the art can be used to construct expression vectorscontaining antibody coding sequences and appropriate transcriptional andtranslational control signals. These methods include, for example, invitro recombinant DNA techniques, synthetic techniques, and in vivogenetic recombination. The invention, thus, provides replicable vectorscomprising a nucleotide sequence encoding an antibody molecule of theinvention, or a heavy or light chain thereof, or a heavy or light chainvariable domain, operably linked to a promoter. Such vectors can includethe nucleotide sequence encoding the constant region of the antibodymolecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO89/01036; and U.S. Pat. No. 5,122,464) and the variable domain of theantibody can be cloned into such a vector for expression of the entireheavy or light chain.

The term “vector” or “expression vector” is used herein to mean vectorsused in accordance with the present invention as a vehicle forintroducing into and expressing a desired gene in a host cell. As knownto those skilled in the art, such vectors can easily be selected fromthe group consisting of plasmids, phages, viruses and retroviruses. Ingeneral, vectors compatible with the instant invention will comprise aselection marker, appropriate restriction sites to facilitate cloning ofthe desired gene and the ability to enter and/or replicate in eukaryoticor prokaryotic cells.

For the purposes of this invention, numerous expression vector systemscan be employed. For example, one class of vector utilizes DNA elementsthat are derived from animal viruses such as bovine papilloma virus,polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses(RSV, MMTV or MOMLV) or SV40 virus. Others involve the use ofpolycistronic systems with internal ribosome binding sites.Additionally, cells that have integrated the DNA into their chromosomescan be selected by introducing one or more markers which allow selectionof transfected host cells. The marker can provide for prototrophy to anauxotrophic host, biocide resistance (e.g., antibiotics) or resistanceto heavy metals such as copper. The selectable marker gene can either bedirectly linked to the DNA sequences to be expressed, or introduced intothe same cell by cotransformation. Additional elements can also beneeded for optimal synthesis of mRNA. These elements can include signalsequences, splice signals, as well as transcriptional promoters,enhancers, and termination signals.

In some embodiments, the cloned variable region genes are inserted intoan expression vector along with the heavy and light chain constantregion genes (e.g., human) synthesized as discussed above. Of course,any expression vector that is capable of eliciting expression ineukaryotic cells can be used in the present invention. Examples ofsuitable vectors include, but are not limited to plasmids pcDNA3,pHCMV/Zeo, pCR3.1, pEF 1/His, pIND/GS, pRc/HCMV2, pSV40/Zeo2,pTRACER-HCMV, pUB6/V5-His, pVAX1, and pZeoSV2 (available fromInvitrogen, San Diego, Calif.), and plasmid pCI (available from Promega,Madison, Wis.). In general, screening large numbers of transformed cellsfor those that express suitably high levels if immunoglobulin heavy andlight chains is routine experimentation that can be carried out, forexample, by robotic systems.

More generally, once the vector or DNA sequence encoding a monomericsubunit of the anti-ILT7 antibody has been prepared, the expressionvector can be introduced into an appropriate host cell. Introduction ofthe plasmid into the host cell can be accomplished by various techniqueswell known to those of skill in the art. These include, but are notlimited to, transfection (including electrophoresis andelectroporation), protoplast fusion, calcium phosphate precipitation,cell fusion with enveloped DNA, microinjection, and infection withintact virus. See, Ridgway (1988) “Mammalian Expression Vectors” inVectors, ed. Rodriguez and Denhardt (Butterworths, Boston, Mass.),Chapter 24.2, pp. 470-472. Typically, plasmid introduction into the hostis via electroporation. The host cells harboring the expressionconstruct are grown under conditions appropriate to the production ofthe light chains and heavy chains, and assayed for heavy and/or lightchain protein synthesis. Exemplary assay techniques includeenzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), orfluorescence-activated cell sorter analysis (FACS), immunohistochemistryand the like.

The expression vector is transferred to a host cell by conventionaltechniques, and the transfected cells are then cultured by conventionaltechniques to produce an antibody for use in the methods describedherein. Thus, the invention includes host cells containing apolynucleotide encoding an antibody of the invention, or a heavy orlight chain thereof, operably linked to a heterologous promoter. In someembodiments for the expression of double-chained antibodies, vectorsencoding both the heavy and light chains can be co-expressed in the hostcell for expression of the entire immunoglobulin molecule, as detailedbelow.

As used herein, “host cells” refers to cells that harbor vectorsconstructed using recombinant DNA techniques and encoding at least oneheterologous gene. In descriptions of processes for isolation ofantibodies from recombinant hosts, the terms “cell” and “cell culture”are used interchangeably to denote the source of antibody unless it isclearly specified otherwise. In other words, recovery of polypeptidefrom the “cells” can mean either from spun down whole cells, or from thecell culture containing both the medium and the suspended cells.

A variety of host-expression vector systems can be utilized to expressantibody molecules for use in the methods described herein. Suchhost-expression systems represent vehicles by which the coding sequencesof interest can be produced and subsequently purified, but alsorepresent cells that can, when transformed or transfected with theappropriate nucleotide coding sequences, express an antibody molecule ofthe invention in situ. These include, but are not limited to,microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformedwith recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expressionvectors containing antibody coding sequences; yeast (e.g.,Saccharomyces, Pichia) transformed with recombinant yeast expressionvectors containing antibody coding sequences; insect cell systemsinfected with recombinant virus expression vectors (e.g., baculovirus)containing antibody coding sequences; plant cell systems infected withrecombinant virus expression vectors (e.g., cauliflower mosaic virus,CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmidexpression vectors (e.g., Ti plasmid) containing antibody codingsequences; or mammalian cell systems (e.g., COS, CHO, BLK, 293, 3T3cells) harboring recombinant expression constructs containing promotersderived from the genome of mammalian cells (e.g., metallothioneinpromoter) or from mammalian viruses (e.g., the adenovirus late promoter;the vaccinia virus 7.5K promoter). Bacterial cells such as Escherichiacoli or eukaryotic cells, especially for the expression of wholerecombinant antibody molecule, are used for the expression of arecombinant antibody molecule. For example, mammalian cells such asChinese hamster ovary cells (CHO), in conjunction with vectorscomprising, e.g., the major intermediate early gene promoter elementfrom human cytomegalovirus are an effective expression system forantibodies (Foecking et al., Gene 45:101 (1986); Cockett et al.,Bio/Technology 8:2 (1990)).

The host cell line used for protein expression is often of mammalianorigin; those skilled in the art are credited with ability to determineparticular host cell lines that are best suited for the desired geneproduct to be expressed therein. Exemplary host cell lines include, butare not limited to, CHO (Chinese Hamster Ovary), DG44 and DUXB11(Chinese Hamster Ovary lines, DHFR minus), HELA (human cervicalcarcinoma), CVI (monkey kidney line), COS (a derivative of CVI with SV40T antigen), VERY, BHK (baby hamster kidney), MDCK, 293, WI38, R1610(Chinese hamster fibroblast) BALBC/3T3 (mouse fibroblast), HAK (hamsterkidney line), SP2/O (mouse myeloma), P3.times.63-Ag3.653 (mousemyeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocyte)and 293 (human kidney). Host cell lines are typically available fromcommercial services, the American Tissue Culture Collection or frompublished literature.

In addition, a host cell strain can be chosen that modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products canbe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells that possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product can be used.

For long-term, high-yield production of recombinant proteins, stableexpression is useful. For example, cell lines that stably express theantibody molecule can be engineered. Rather than using expressionvectors that contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells can beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method canadvantageously be used to engineer cell lines which stably express theantibody molecule.

A number of selection systems can be used, including, but not limitedto, the herpes simplex virus thymidine kinase (Wigler et al., Cell11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase(Szybalska and Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), andadenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980))genes can be employed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl.Acad Sci. USA 78:1527 (1981)); gpt, which confers resistance tomycophenolic acid (Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78:2072(1981)); neo, which confers resistance to the aminoglycoside G-418Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991);Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan,Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem.62:191-217 (1993); TIB TECH 11(5):155-215 (May, 1993); and hygro, whichconfers resistance to hygromycin (Santerre et al., Gene 30:147 (1984).Methods commonly known in the art of recombinant DNA technology whichcan be used are described in Ausubel et al. (1993) Current Protocols inMolecular Biology (John Wiley & Sons, NY); Kriegler (1990) “GeneTransfer and Expression” in A Laboratory Manual (Stockton Press, NY);Dracopoli et al. (eds) (1994) Current Protocols in Human Genetics (JohnWiley & Sons, NY) Chapters 12 and 13; Colberre-Garapin et al. (1981) J.Mol. Biol. 150:1, which are incorporated by reference herein in theirentireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington and Hentschel (1987) “TheUse of Vectors Based on Gene Amplification for the Expression of ClonedGenes in Mammalian Cells in DNA Cloning” (Academic Press, NY) Vol. 3.When a marker in the vector system expressing antibody is amplifiable,increase in the level of inhibitor present in culture of host cell willincrease the number of copies of the marker gene. Since the amplifiedregion is associated with the antibody gene, production of the antibodywill also increase (Crouse et al., Mol. Cell. Biol. 3:257 (1983)).

In vitro production allows scale-up to give large amounts of the desiredpolypeptides. Techniques for mammalian cell cultivation under tissueculture conditions are known in the art and include homogeneoussuspension culture, e.g. in an airlift reactor or in a continuousstirrer reactor, or immobilized or entrapped cell culture, e.g. inhollow fibers, microcapsules, on agarose microbeads or ceramiccartridges. If necessary and/or desired, the solutions of polypeptidescan be purified by the customary chromatography methods, for example gelfiltration, ion-exchange chromatography, chromatography overDEAE-cellulose or (immuno-)affinity chromatography, e.g., afterpreferential biosynthesis of a synthetic hinge region polypeptide orprior to or subsequent to the HIC chromatography step described herein.

Genes encoding anti-ILT7 antibodies, or antigen-binding fragments,variants, or derivatives thereof of the invention can also be expressedin non-mammalian cells such as insect, bacteria or yeast or plant cells.Bacteria that readily take up nucleic acids include members of theenterobacteriaceae, such as strains of Escherichia coli or Salmonella;Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus, andHaemophilus influenzae. It will further be appreciated that, whenexpressed in bacteria, the heterologous polypeptides typically becomepart of inclusion bodies. The heterologous polypeptides must beisolated, purified and then assembled into functional molecules. Wheretetravalent forms of antibodies are desired, the subunits will thenself-assemble into tetravalent antibodies (WO 02/096948A2).

In bacterial systems, a number of expression vectors can beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified can be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791(1983)), in which the antibody coding sequence can be ligatedindividually into the vector in frame with the lacZ coding region sothat a fusion protein is produced; pIN vectors (Inouye and Inouye,Nucleic Acids Res. 13:3101-3109 (1985); Van Heeke and Schuster, J. Biol.Chem. 24:5503-5509 (1989)); and the like. pGEX vectors can also be usedto express foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding to amatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In addition to prokaryotes, eukaryotic microbes can also be used.Saccharomyces cerevisiae, or common baker's yeast, is the most commonlyused among eukaryotic microorganisms although a number of other strainsare commonly available, e.g., Pichia pastoris.

For expression in Saccharomyces, the plasmid YRp7, for example,(Stinchcomb et al., Nature 282:39 (1979); Kingsman et al., Gene 7:141(1979); Tschemper et al., Gene 10:157 (1980)) is commonly used. Thisplasmid already contains the TRP1 gene, which provides a selectionmarker for a mutant strain of yeast lacking the ability to grow intryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, Genetics 85:12(1977)). The presence of the trp1 lesion as a characteristic of theyeast host cell genome then provides an effective environment fordetecting transformation by growth in the absence of tryptophan.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is typically used as a vector to express foreign genes. Thevirus grows in Spodoptera frugiperda cells. The antibody coding sequencecan be cloned individually into non-essential regions (for example thepolyhedrin gene) of the virus and placed under control of an AcNPVpromoter (for example the polyhedrin promoter).

Once a binding molecule of the invention has been recombinantlyexpressed, it can be purified by any method known in the art forpurification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigen after Protein A, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins.Alternatively, a beneficial method for increasing the affinity ofantibodies of the invention is disclosed in U.S. Patent ApplicationPublication No. 2002 0123057 A1.

VII. Treatment Methods Using Therapeutic Anti-ILT7 Binding Molecules

Methods of the invention are directed to the use of anti-ILT7 bindingmolecules, e.g., antibodies, including antigen-binding fragments,variants, and derivatives thereof, to treat patients having a diseaseassociated with ILT7 expression or ILT7-expressing cells. By“ILT7-expressing cell” is intended cells expressing ILT7 antigen.Methods for detecting ILT7 expression in cells are well known in the artand include, but are not limited to, PCR techniques,immunohistochemistry, flow cytometry, Western blot, ELISA, and the like.

Though the following discussion refers to diagnostic methods andtreatment of various diseases and disorders with an anti-ILT7 antibodyof the invention, the methods described herein are also applicable tothe antigen-binding fragments, variants, and derivatives of theseanti-ILT7 antibodies that retain the desired properties of the anti-ILT7antibodies of the invention, e.g., capable of specifically binding ILT7and neutralizing ILT7 pathogenic activity.

In one embodiment, treatment includes the application or administrationof an anti-ILT7 binding molecule, e.g., an antibody or antigen bindingfragment, variant, or derivative thereof of the current invention to asubject or patient, or application or administration of the anti-ILT7binding molecule to an isolated tissue or cell line from a subject orpatient, where the subject or patient has a disease, a symptom of adisease, or a predisposition toward a disease. In another embodiment,treatment is also intended to include the application or administrationof a pharmaceutical composition comprising the anti-ILT7 bindingmolecule, e.g., an antibody or antigen binding fragment, variant, orderivative thereof of the current invention to a subject or patient, orapplication or administration of a pharmaceutical composition comprisingthe anti-ILT7 binding molecule to an isolated tissue or cell line from asubject or patient, who has a disease, a symptom of a disease, or apredisposition toward a disease.

The anti-ILT7 binding molecules, e.g., antibodies or antigen-bindingfragments, variants, or derivatives thereof of the present invention areuseful for the treatment of various autoimmune conditions. For example,therapy with at least one anti-ILT7 antibody causes a physiologicalresponse, for example, a reduction in interferon, that is beneficialwith respect to treatment of disease states associated withILT7-expressing cells in a human.

In one embodiment, the invention relates to anti-ILT7 binding molecules,e.g., antibodies or antigen-binding fragments, variants, or derivativesthereof for use as a medicament, in particular for use in the treatmentor prophylaxis of an autoimmune condition or disease. Examples ofautoimmune diseases include, but are not limited to: myositis, diabetes,Hashimoto's disease, autoimmune adrenal insufficiency, pure red cellanemia, multiple sclerosis, rheumatoid carditis, systemic lupuserythematosus, psoriasis, rheumatoid arthritis, chronic inflammation,Sjogren's syndrome, polymyositis, dermatomyositis, inclusion bodymyositis, juvenile myositis, and scleroderma.

In accordance with the methods of the present invention, at least oneanti-ILT7 binding molecule, e.g., an antibody or antigen bindingfragment, variant, or derivative thereof as defined elsewhere herein isused to promote a positive therapeutic response with respect to anautoimmune response. By “positive therapeutic response” with respect toautoimmune treatment is intended an improvement in the disease inassociation with the activity of these binding molecules, e.g.,antibodies or antigen-binding fragments, variants, or derivativesthereof, and/or an improvement in the symptoms associated with thedisease. That is, a decrease in interferon-alpha levels, a decrease inthe number or activity of plasmacytoid dendritic cells, or a decrease inone or more symptoms associated with the disease can be observed. Thus,for example, an improvement in the disease can be characterized as acomplete response. By “complete response” is intended an absence ofclinically detectable disease with normalization of any previously testresults. Such a response must persist for at least one month followingtreatment according to the methods of the invention. Alternatively, animprovement in the disease can be categorized as being a partialresponse.

The anti-ILT7 binding molecules, e.g., antibodies or antigen bindingfragments, variants, or derivatives thereof described herein can alsofind use in the treatment of autoimmune diseases and deficiencies ordisorders of the immune system that are associated with ILT7 expressingcells. Autoimmune diseases are characterized by cellular, tissue and/ororgan injury caused by an immunologic reaction of the subject to its owncells, tissues and/or organs. In one embodiment, the autoimmune diseaseis systemic lupus erythematosus.

Clinical response can be assessed using screening techniques such asmagnetic resonance imaging (MRI) scan, x-radiographic imaging, computedtomographic (CT) scan, flow cytometry or fluorescence-activated cellsorter (FACS) analysis, histology, gross pathology, and blood chemistry,including but not limited to changes detectable by ELISA, RIA,chromatography, and the like. In addition to these positive therapeuticresponses, the subject undergoing therapy with the anti-ILT7 bindingmolecule, e.g., an antibody or antigen-binding fragment, variant, orderivative thereof, can experience the beneficial effect of animprovement in the symptoms associated with the disease.

A further embodiment of the invention is the use of anti-ILT7 bindingmolecules, e.g., antibodies or antigen-binding fragments, variants, orderivatives thereof, for diagnostic monitoring of protein levels intissue as part of a clinical testing procedure, e.g., to determine theefficacy of a given treatment regimen. For example, detection can befacilitated by coupling the antibody to a detectable substance. Examplesof detectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,and radioactive materials. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S, or ³H.

VIII. Pharmaceutical Compositions and Administration Methods

Methods of preparing and administering anti-ILT7 binding molecules,e.g., antibodies, or antigen-binding fragments, variants, or derivativesthereof, provided herein to a subject in need thereof are well known toor are readily determined by those skilled in the art.

As discussed herein, anti-ILT7 binding molecules, e.g., antibodies, orantigen-binding fragments, variants, or derivatives thereof, providedherein can be administered in a pharmaceutically effective amount forthe in vivo treatment of ILT7-expressing cell-mediated diseases such ascertain types autoimmune diseases. In this regard, it will beappreciated that the disclosed binding molecules of the invention willbe formulated so as to facilitate administration and promote stabilityof the active agent. Pharmaceutical compositions in accordance with thepresent invention can comprise a pharmaceutically acceptable, non-toxic,sterile carrier. For the purposes of the instant application, apharmaceutically effective amount of an anti-ILT7 binding molecule,e.g., an antibody, or antigen-binding fragment, variant, or derivativethereof, conjugated or unconjugated, shall be held to mean an amountsufficient to achieve effective binding to a target and to achieve abenefit, e.g., to ameliorate symptoms of a disease or condition or todetect a substance or a cell.

Pharmaceutical compositions suitable for injectable should be sterileand should be fluid to the extent that easy syringability exists. Itshould be stable under the conditions of manufacture and storage andwill beneficially be preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents. Suitable formulations for use in the therapeutic methodsdisclosed herein are described in Remington's Pharmaceutical Sciences(Mack Publishing Co.) 16th ed. (1980).

In keeping with the scope of the present disclosure, anti-ILT7antibodies, or antigen-binding fragments, variants, or derivativesthereof of the invention can be administered to a human or other animalin accordance with the aforementioned methods of treatment in an amountsufficient to produce a therapeutic effect. The anti-ILT7 antibodies, orantigen-binding fragments, variants, or derivatives thereof of theinvention can be administered to such human or other animal in aconventional dosage form prepared by combining the antibody orantigen-binding fragment, variant, or derivative thereof of theinvention with a conventional pharmaceutically acceptable carrier ordiluent according to known techniques. It will be recognized by one ofskill in the art that the form and character of the pharmaceuticallyacceptable carrier or diluent is dictated by the amount of activeingredient with which it is to be combined, the route of administrationand other well-known variables. Those skilled in the art will furtherappreciate that a cocktail comprising one or more species of anti-ILT7binding molecules, e.g., antibodies, or antigen-binding fragments,variants, or derivatives thereof, of the invention can prove to beparticularly effective.

By “therapeutically effective dose or amount” or “effective amount” isintended an amount of anti-ILT7 binding molecule, e.g., antibody orantigen binding fragment, variant, or derivative thereof, that whenadministered brings about a positive therapeutic response with respectto treatment of a patient with a disease or condition to be treated.

Therapeutically effective doses of the compositions of the presentinvention, for treatment of ILT7-expressing cell-mediated diseases suchas certain types of autoimmune diseases including e.g., systemic lupuserythematosus, vary depending upon many different factors, includingmeans of administration, target site, physiological state of thepatient, whether the patient is human or an animal, other medicationsadministered, and whether treatment is prophylactic or therapeutic.Usually, the patient is a human, but non-human mammals includingtransgenic mammals can also be treated. Treatment dosages can betitrated to optimize safety and efficacy.

The present invention also provides for the use of an anti-ILT7 bindingmolecule, e.g., an antibody or antigen-binding fragment, variant, orderivative thereof, in the manufacture of a medicament for treating anautoimmune disease, including, e.g., systemic lupus erythematosus.

IX. Diagnostics

The invention further provides a diagnostic method useful duringdiagnosis of ILT7-expressing cell-mediated diseases such as certaintypes of autoimmune diseases including, e.g., systemic lupuserythematosus, which involves measuring the expression level of ILT7protein or transcript in tissue or other cells or body fluid from anindividual and comparing the measured expression level with a standardILT7 expression level in normal tissue or body fluid, whereby anincrease in the expression level compared to the standard is indicativeof a disorder.

The anti-ILT7 antibodies of the invention and antigen-binding fragments,variants, and derivatives thereof, can be used to assay ILT7 proteinlevels in a biological sample using classical immunohistological methodsknown to those of skill in the art (e.g., see Jalkanen, et al., J. Cell.Biol. 101:976-985 (1985); Jalkanen et al., J. Cell Biol. 105:3087-3096(1987)). Other antibody-based methods useful for detecting ILT7 proteinexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA), immunoprecipitation, or Western blotting. Suitable assaysare described in more detail elsewhere herein.

By “assaying the expression level of ILT7 polypeptide” is intendedqualitatively or quantitatively measuring or estimating the level ofILT7 polypeptide in a first biological sample either directly (e.g., bydetermining or estimating absolute protein level) or relatively (e.g.,by comparing to the disease associated polypeptide level in a secondbiological sample). ILT7 polypeptide expression level in the firstbiological sample can be measured or estimated and compared to astandard ILT7 polypeptide level, the standard being taken from a secondbiological sample obtained from an individual not having the disorder orbeing determined by averaging levels from a population of individualsnot having the disorder. As will be appreciated in the art, once the“standard” ILT7 polypeptide level is known, it can be used repeatedly asa standard for comparison.

By “biological sample” is intended any biological sample obtained froman individual, cell line, tissue culture, or other source of cellspotentially expressing ILT7. Methods for obtaining tissue biopsies andbody fluids from mammals are well known in the art.

X. Immunoassays

Anti-ILT7 binding molecules, e.g., antibodies, or antigen-bindingfragments, variants, or derivatives thereof of the invention can beassayed for immunospecific binding by any method known in the art. Theimmunoassays that can be used include but are not limited to competitiveand non-competitive assay systems using techniques such as Westernblots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay),“sandwich” immunoassays, immunoprecipitation assays, precipitinreactions, gel diffusion precipitin reactions, immunodiffusion assays,agglutination assays, complement-fixation assays, immunoradiometricassays, fluorescent immunoassays, protein A immunoassays, to name but afew. Such assays are routine and well known in the art (see, e.g.,Ausubel et al., eds, (1994) Current Protocols in Molecular Biology (JohnWiley & Sons, Inc., NY) Vol. 1, which is incorporated by referenceherein in its entirety). Exemplary immunoassays are described brieflybelow (but are not intended by way of limitation).

Anti-ILT7 antibodies, or antigen-binding fragments, variants, orderivatives thereof of the invention, additionally, can be employedhistologically, as in immunofluorescence, immunoelectron microscopy ornon-immunological assays, for in situ detection of ILT7 protein orconserved variants or peptide fragments thereof. In situ detection canbe accomplished by removing a histological specimen from a patient, andapplying thereto a labeled anti-ILT7 antibody, or antigen-bindingfragment, variant, or derivative thereof, e.g., applied by overlayingthe labeled antibody (or fragment) onto a biological sample. Through theuse of such a procedure, it is possible to determine not only thepresence of ILT7 protein, or conserved variants or peptide fragments,but also its distribution in the examined tissue. Using the presentinvention, those of ordinary skill will readily perceive that any of awide variety of histological methods (such as staining procedures) canbe modified in order to achieve such in situ detection.

Immunoassays and non-immunoassays for ILT7 gene products or conservedvariants or peptide fragments thereof will typically comprise incubatinga sample, such as a biological fluid, a tissue extract, freshlyharvested cells, or lysates of cells which have been incubated in cellculture, in the presence of a detectably labeled antibody capable ofbinding to ILT7 or conserved variants or peptide fragments thereof, anddetecting the bound antibody by any of a number of techniques well knownin the art.

The biological sample can be brought in contact with and immobilizedonto a solid phase support or carrier such as nitrocellulose, or othersolid support which is capable of immobilizing cells, cell particles orsoluble proteins. The support can then be washed with suitable buffersfollowed by treatment with the detectably labeled anti-ILT7 antibody, orantigen-binding fragment, variant, or derivative thereof. The solidphase support can then be washed with the buffer a second time to removeunbound antibody. Optionally the antibody is subsequently labeled. Theamount of bound label on solid support can then be detected byconventional means.

By “solid phase support or carrier” is intended any support capable ofbinding an antigen or an antibody. Well-known supports or carriersinclude glass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, gabbros, andmagnetite. The nature of the carrier can be either soluble to someextent or insoluble for the purposes of the present invention. Thesupport material can have virtually any possible structuralconfiguration so long as the coupled molecule is capable of binding toan antigen or antibody. Thus, the support configuration can bespherical, as in a bead, or cylindrical, as in the inside surface of atest tube, or the external surface of a rod. Alternatively, the surfacecan be flat such as a sheet, test strip, etc. Exemplary supports includepolystyrene beads. Those skilled in the art will know many othersuitable carriers for binding antibody or antigen, or will be able toascertain the same by use of routine experimentation.

The binding activity of a given lot of anti-ILT7 antibody, orantigen-binding fragment, variant, or derivative thereof can bedetermined according to well known methods. Those skilled in the artwill be able to determine operative and optimal assay conditions foreach determination by employing routine experimentation.

The binding affinity of an antibody to an antigen and the off-rate of anantibody-antigen interaction can be determined by competitive bindingassays. One example of a competitive binding assay is a radioimmunoassaycomprising the incubation of labeled antigen (e.g., ³H or ¹²⁵I) with theantibody of interest in the presence of increasing amounts of unlabeledantigen, and the detection of the antibody bound to the labeled antigen.The affinity of the antibody of interest for a particular antigen andthe binding off-rates can be determined from the data by scatchard plotanalysis. Competition with a second antibody can also be determinedusing radioimmunoassays. In this case, the antigen is incubated withantibody of interest is conjugated to a labeled compound (e.g., ³H or¹²⁵I) in the presence of increasing amounts of an unlabeled secondantibody.

There are a variety of methods available for measuring the affinity ofan antibody-antigen interaction, but relatively few for determining rateconstants. Most of the methods rely on either labeling antibody orantigen, which inevitably complicates routine measurements andintroduces uncertainties in the measured quantities.

Surface plasmon reasonance (SPR) as performed on BIACORE® offers anumber of advantages over conventional methods of measuring the affinityof antibody-antigen interactions: (i) no requirement to label eitherantibody or antigen; (ii) antibodies do not need to be purified inadvance, cell culture supernatant can be used directly; (iii) real-timemeasurements, allowing rapid semi-quantitative comparison of differentmonoclonal antibody interactions, are enabled and are sufficient formany evaluation purposes; (iv) biospecific surface can be regenerated sothat a series of different monoclonal antibodies can easily be comparedunder identical conditions; (v) analytical procedures are fullyautomated, and extensive series of measurements can be performed withoutuser intervention. BIAapplications Handbook, version AB (reprinted1998), BIACORE® code No. BR-1001-86; BIAtechnology Handbook, version AB(reprinted 1998), BIACORE® code No. BR-1001-84. SPR based bindingstudies require that one member of a binding pair be immobilized on asensor surface. The binding partner immobilized is referred to as theligand. The binding partner in solution is referred to as the analyte.In some cases, the ligand is attached indirectly to the surface throughbinding to another immobilized molecule, which is referred as thecapturing molecule. SPR response reflects a change in mass concentrationat the detector surface as analytes bind or dissociate.

Based on SPR, real-time BIACORE® measurements monitor interactionsdirectly as they happen. The technique is well suited to determinationof kinetic parameters. Comparative affinity ranking is simple toperform, and both kinetic and affinity constants can be derived from thesensorgram data.

When analyte is injected in a discrete pulse across a ligand surface,the resulting sensorgram can be divided into three essential phases: (i)Association of analyte with ligand during sample injection; (ii)Equilibrium or steady state during sample injection, where the rate ofanalyte binding is balanced by dissociation from the complex; (iii)Dissociation of analyte from the surface during buffer flow.

The association and dissociation phases provide information on thekinetics of analyte-ligand interaction (k_(a) and k_(d), the rates ofcomplex formation and dissociation, k_(d)/k_(a)=K_(D)). The equilibriumphase provides information on the affinity of the analyte-ligandinteraction (K_(D)).

BIAevaluation software provides comprehensive facilities for curvefitting using both numerical integration and global fitting algorithms.With suitable analysis of the data, separate rate and affinity constantsfor interaction can be obtained from simple BIACORE® investigations. Therange of affinities measurable by this technique is very broad rangingfrom mM to pM.

Epitope specificity is an important characteristic of a monoclonalantibody. Epitope mapping with BIACORE®, in contrast to conventionaltechniques using radioimmunoassay, ELISA or other surface adsorptionmethods, does not require labeling or purified antibodies, and allowsmulti-site specificity tests using a sequence of several monoclonalantibodies. Additionally, large numbers of analyses can be processedautomatically.

Pair-wise binding experiments test the ability of two MAbs to bindsimultaneously to the same antigen. MAbs directed against separateepitopes will bind independently, whereas MAbs directed againstidentical or closely related epitopes will interfere with each other'sbinding. These binding experiments with BIACORE® are straightforward tocarry out.

For example, one can use a capture molecule to bind the first Mab,followed by addition of antigen and second MAb sequentially. Thesensorgrams will reveal: (1) how much of the antigen binds to first Mab,(2) to what extent the second MAb binds to the surface-attached antigen,(3) if the second MAb does not bind, whether reversing the order of thepair-wise test alters the results.

Peptide inhibition is another technique used for epitope mapping. Thismethod can complement pair-wise antibody binding studies, and can relatefunctional epitopes to structural features when the primary sequence ofthe antigen is known. Peptides or antigen fragments are tested forinhibition of binding of different MAbs to immobilized antigen. Peptidesthat interfere with binding of a given MAb are assumed to bestructurally related to the epitope defined by that MAb.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. See, for example, Sambrook etal., ed. (1989) Molecular Cloning A Laboratory Manual (2nd ed.; ColdSpring Harbor Laboratory Press); Sambrook et al., ed. (1992) MolecularCloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); D.N. Glover ed., (1985) DNA Cloning, Volumes I and II; Gait, ed. (1984)Oligonucleotide Synthesis; Mullis et al. U.S. Pat. No. 4,683,195; Hamesand Higgins, eds. (1984) Nucleic Acid Hybridization; Hames and Higgins,eds. (1984) Transcription And Translation; Freshney (1987) Culture OfAnimal Cells (Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRLPress) (1986); Perbal (1984) A Practical Guide To Molecular Cloning; thetreatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Miller andCalos eds. (1987) Gene Transfer Vectors For Mammalian Cells, (ColdSpring Harbor Laboratory); Wu et al., eds., Methods In Enzymology, Vols.154 and 155; Mayer and Walker, eds. (1987) Immunochemical Methods InCell And Molecular Biology (Academic Press, London); Weir and Blackwell,eds., (1986) Handbook Of Experimental Immunology, Volumes I-IV;Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., (1986); and in Ausubel et al. (1989) CurrentProtocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.).

General principles of antibody engineering are set forth in Borrebaeck,ed. (1995) Antibody Engineering (2nd ed.; Oxford Univ. Press). Generalprinciples of protein engineering are set forth in Rickwood et al., eds.(1995) Protein Engineering, A Practical Approach (IRL Press at OxfordUniv. Press, Oxford, Eng.). General principles of antibodies andantibody-hapten binding are set forth in: Nisonoff (1984) MolecularImmunology (2nd ed.; Sinauer Associates, Sunderland, Mass.); and Steward(1984) Antibodies, Their Structure and Function (Chapman and Hall, NewYork, N.Y.). Additionally, standard methods in immunology known in theart and not specifically described are generally followed as in CurrentProtocols in Immunology, John Wiley & Sons, New York; Stites et al.,eds. (1994) Basic and Clinical Immunology (8th ed; Appleton & Lange,Norwalk, Conn.) and Mishell and Shiigi (eds) (1980) Selected Methods inCellular Immunology (W.H. Freeman and Co., NY).

Standard reference works setting forth general principles of immunologyinclude Current Protocols in Immunology, John Wiley & Sons, New York;Klein (1982) J., Immunology: The Science of Self-Nonself Discrimination(John Wiley & Sons, NY); Kennett et al., eds. (1980) MonoclonalAntibodies, Hybridoma: A New Dimension in Biological Analyses (PlenumPress, NY); Campbell (1984) “Monoclonal Antibody Technology” inLaboratory Techniques in Biochemistry and Molecular Biology, ed. Burdenet al., (Elsevere, Amsterdam); Goldsby et al., eds. (2000) KubyImmunology (4th ed.; H. Freemand & Co.); Roitt et al. (2001) Immunology(6th ed.; London: Mosby); Abbas et al. (2005) Cellular and MolecularImmunology (5th ed.; Elsevier Health Sciences Division); Kontermann andDubel (2001) Antibody Engineering (Springer Verlan); Sambrook andRussell (2001) Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Press); Lewin (2003) Genes VIII (Prentice Hall2003); Harlow andLane (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor Press);Dieffenbach and Dveksler (2003) PCR Primer (Cold Spring Harbor Press).

All of the references cited above, as well as all references citedherein, are incorporated herein by reference in their entireties.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Materials and Methods

Biological Samples

Human peripheral blood from normal healthy volunteers was obtainedthrough MedImmune Blood Donor Program, with written informed consent andapproval from the IRB. Peripheral blood mononuclear cells (PBMC) wereisolated from fresh whole blood using Vacutainer CPT cell preparationtubes with sodium citrate (Becton Dickinson Biosciences, NJ, USA). Tubeswere spun at 17000 g for 25 min, 22° C., with minimal braking. After thespin, the serum was removed, and the cellular buffy coat was transferredto conical 50 mL tubes (BD Biosciences). Purified cells were washedtwice with sterile phosphate buffered saline (PBS) (Invitrogen LifeTechnologies, CA, USA) at 350 g for 10 minutes at 22° C. Cells wereresuspended in PBS or RPMI 1640 media supplemented with 10% fetal bovineserum (Invitrogen) and were filtered using BD Falcon 5 mL tubes withcell strainer caps (BD Biosciences). Cell densities were determinedusing a Vi-Cell XR® cell counter (Beckman Coulter, Calif., USA).

Cynomolgus peripheral blood from healthy animals was obtained fromBioqual (Bioqual, Inc. MD, USA), in accordance with the guidelines ofthe National Institutes of Health for care and use of primates.Cynomolgus PBMCs were either isolated using Vacutainer CPT cellpreparation tubes with sodium citrate (as described above) or withHistopaque 10771 (Sigma-Aldrich, Mo., USA). Briefly, fresh whole bloodwas adjusted to 50× the initial blood volume with sterile PBS. Then, 25mL of diluted blood was overlaid onto 10 mL of 90% Histopaque 10771(Sigma Aldrich) and samples were spun at 400 g for 20 min at roomtemperature with minimal braking. Cellular disc was removed andtransferred to a new 50 mL conical tube. Purified cells were washedtwice with sterile PBS at 350 g for 10 minutes at 22° C. Cells wereresuspended in PBS or RPMI 1640 media supplemented with 10% fetal bovineserum, filtered, and counted as described above.

Cells

CT-125 and CT-550 cells were obtained from Dr. Yong-Jun Liu (Universityof Texas M. D. Anderson Cancer Center, Houston, Tex., USA). CT-125 cellswere generated by transducing 2B4 murine T-cell hybridoma with untaggedmouse FcεR1γ and a NFAT-GFP reporter gene and CT-550 cells weregenerated by transducing CT-125 cells with HA-tagged human ILT7 (OhtsukaM. et al., PNAS 101: 8126-8131 (2004); Cao W. et al., JEM 203: pp1399-1405 (2006)). CT-125 Cyno ILT7 stable cell line was generated bytransfecting CT-125 cells with cynomolgus monkey ILT7 gene cloned into apME18X plasmid vector. CT cells were cultured in RPMI 1640 supplementedwith 10% fetal bovine serum (FBS) and 1× penicillin/streptomycin (allfrom Invitrogen Life Technologies).

KC1333 cells were obtained from Biowa (Biowa, NJ, USA). KC1333 cellswere cultured in Advance RPMI 1640 supplemented with 10% FBS, 4 mML-Glutamine, 0.2 μg/mL Geneticin (all from Invitrogen), and 18.3 pg/mLof recombinant human IL-2 (PeproTech, NJ, USA).

Antibodies and Reagents

Anti-ILT7 humanized antibody variants, anti-ILT7 clone 7C7 (7C7), andhumanized isotype control R347 were generated at MedImmune.Allophycocyanin (APC) conjugated anti-ILT7 humanized antibody variants,7C7, and isotype control R347 were generated using APC monoclonalantibody labeling kits (Thermo Fisher Scientific, IL, USA).R-Phycoerythrin (PE) and FITC-labeled anti-human BDCA-2 antibody (cloneAC144), R-PE anti-human BDCA-4 (clone AD5-17F6), and Human FcR BlockingReagent were from Miltenyi Biotech, CA, USA. Anti-human CD123 (clone7G3) conjugated to either R-PE, FITC or APC, Alexa Fluor 488 anti-humanCD8 (clone RPA-T8), Alexa Fluor 488 anti-human CD3 (clone SP34-2), FITCanti-human CD14 (clone M5E2), FITC anti-human CD20 (clone 2H7) andPerCP-Cy 5.5 anti-human HLA-DR (clone G46-6) were obtained from BDBiosciences. Pacific Blue anti-human CD56 antibody (clone MEM-188) wasobtained from BioLegend, CA, USA. DyLight 649-labeled anti-human IgG andhuman whole IgG were from Jackson Immunoresearch, PA, USA.

Whole blood staining was done using BD FACS Lysing Solution (BDBiosciences). 7-AAD was obtained from Invitrogen. Human male AB plasmawas from Sigma-Aldrich. Recombinant human IL-2 was from R&D Systems, MN,USA, and recombinant human interferon β (IFN-β) was from PBL Biomedical,NJ, USA. CpG A ODN 2216 was from InvivoGen, CA, USA.

Labeling of Human and Cynomolgus Recombinant IL 77

Proteins were biotinylated via free amines using EZ linkSulfo-NHS-LC-Biotin (Thermo/Pierce, product: 21335). The reagent wasdissolved in anhydrous dimethylformamide, and the PBS based proteinsolutions were adjusted to pH ˜8 with 1 M NaHCO₃ in D-PBS.

Label incorporations were assessed by MALDI-TOF mass spectrometry in allcases, and unreacted reagents were cleared by buffer exchange usingD-PBS equilibrated disposable Sephadex G25 columns. For biotinylationsthe final protein concentrations were determined by 280 nm absorbanceusing extinction coefficients calculated from amino acid sequences.

ELISA Binding Assay

Single-chain Fv fragments were displayed on phage particles and testedin a binding assay to determine cross-reactivity and specificity to apanel of recombinant antigens. Phage-displayed scFv supernatant sampleswere generated in 96-well deep well plates as follows. 5 μl of culturefrom each well of a 96-well master plate was transferred into a Greinerdeep well culture plate containing 500 μl of 2TYAG (2TY+100 μg/mlampicillin+2% glucose) media and incubated for 5 hours at 37° C., 280rpm. K07 M13 helper phage (diluted to 1.5×10¹¹ pfu/ml in 2TYAG) was thenadded at 100 μl/well and the plate incubated at 37° C., 150 rpm to allowinfection. The plate was spun down at 3200 rpm for 10 minutes and thesupernatant removed. Bacterial pellets were resuspended in 500 μl/well2TYAK (2TY+100 μg/ml ampicillin+50 μg/ml kanamycin) and the plateincubated overnight at 25° C., 280 rpm. In the morning, 500 μl of 6%(w/v) skimmed milk powder in 2×PBS was added to each well and the plateincubated for 1 hour at room temperature. The plate was then centrifugedat 3200 rpm for 10 minutes and the blocked phage-displayed scFvsupernatants were used directly in ELISA experiments.

For EC50 determinations, typically purified IgGs were diluted 3-fold in3% (w/v) dried-milk powder in PBS (PBS-M), to give 11 concentrationpoints. 96-well Greiner polypropylene plates (Greiner, 650201) were usedfor dilution preparation. Generally, each dilution was prepared induplicate. IgG dilutions were allowed to block in PBS-M for 1 hour atroom temperature before being used directly in ELISA experiments.

The IL-T7 binding assays were plate-based ELISAs performed essentiallyas follows. Not all antigens were used in every experiment, buttypically a human, a mouse, and a cynomolgus IL-T7 antigen was tested.Relevant control antigens (bovine insulin plus IL-4Rα FLAG®His, ifappropriate) were also used to test for non-specific binding. With theexception of bovine insulin, all antigens were biotinylated and all weregenerated using bacterial expression. IL-T7 antigens were biotinylatedvia free sulfhydryl groups using EZ link Biotin-BMCC (Perbio/Pierce21900). The method for generation of IL-4Rα FLAG® His, which was used asa control antigen, is described in WO/2010/070346. IL-4Rα FLAG®His wasbiotinylated via free amines using EZ link Sulfo-NHS-LC-Biotin(Perbio/Pierce, 21335).

Streptavidin plates (Thermo Scientific, AB-1226) were coated withbiotinylated antigen at 0.5 μg/ml in PBS and incubated overnight at 4°C. Plates were washed 3× with PBS and blocked with 300 μl/well blockingbuffer (PBS-M) for 1 hour. Plates were washed 1× with PBS and blockedsamples added, 50 μl/well for 1 hour at room temperature. Plates werewashed 3× with PBS-T (PBS+1% (v/v) Tween-20) and detection reagents[anti-human IgG HRP (Sigma, A0170) or anti-M13-HRP antibody (Amersham,27-9421-01) for detection of IgG or phage-displayed scFv, respectively]at 1:5000 dilutions were added at 50 μl/well in PBS-M for 1 hour at roomtemperature. Plates were washed 3× with PBS-T and developed with TMB, 50μl/well (Sigma, T0440). The reaction was quenched with 50 μl/well 0.1MH₂SO₄ before reading on an EnVision™ plate reader, or similar equipment,at 450 nm.

Dose response curves were plotted for IgG titrations using Prism(Graphpad) curve-fitting software. Phage-displayed scFv were consideredto bind the IL-T7 antigen if the absorbance 450 nm was >0.5, and<0.1-0.2 for the same sample on controls (insulin and IL-4Rα Flag®His).Single-chain Fv fragments were displayed on phage particles and testedas unpurified preparations in a single point ELISA screen.

Fluorescence Microvolume Assay Technology (FMAT) Cell Binding Assay

This homogeneous assay assessed the binding of crude scFv supernatantsamples or purified IgG to Chinese Hamster Ovary (CHO) cells expressingeither human or cynomolgus ILT7 in a 384-well (Costar 3655) format. ScFvor Ab binding to cells was detected using a mouse anti-His/goatanti-mouse Alexafluor®-647 labelled antibody (Molecular Probes A21236)mix, or goat anti-human Alexafluor®-647 labelled antibody (MolecularProbes A21445) respectively. Plates were read on the Applied BiosystemsCellular Detection system 8200 reader. The Helium neon excitation laserwas focused within 100 μm depth of the bottom of the well, scanning anarea 1 mm². The cells settled at the bottom of the well, and upon laserexcitation at 633 nm, those beads with fluorophore bound (where thelocal concentration of fluorophore is relatively high compared tounbound fluorophore) emitted a signal at 650-685 nm that was measuredusing photomultiplier tube-1 (PMT1). Unbound fluorophore in solution wasoutside the excitation depth or at a relatively low local concentration,and thus did not emit a significant signal. The presence of scFv or IgGsamples binding to the cells at the bottom of the well caused anincrease in Alexafluor-labeled detection antibody within the excitationdepth. This was measured as an increase in fluorescence.

In these experiments, the assay buffer was PBS (Gibco 14190-094)containing 0.1% BSA (Sigma A9576-50 ml), 0.1% Tween-20 (Sigma P2287),and 0.01% sodium azide. To create the ScFv detection mix, mouse anti-Hisand anti-mouse AF647 antibodies were mixed at 1 ug/ml and 2 ug/mlrespectively in assay buffer. To create the IgG detection mix,anti-human AF647 antibody was prepared at 2 ug/ml in assay buffer.

The cells used were CHO-K1 cells expressing either human or cynomolgusILT7 that were cultured using standard tissue culture techniques. Cellswere grown to approximately 80% confluence in F-10 (Gibco,22390-025)+10% FCS (SAFC Biosciences, 13068C)+0.5 mg/ml Zeocin(Invitrogen, R250-01), washed with PBS, detached with accutase (PAA,L11-007), and resuspended in PBS at 1.5×10 cells/ml.

Crude scFv supernatant samples were generated in 96 deep well plates. A5 μl culture from each well of a 96-well master plate was transferredinto a Greiner deep well culture plate containing 900 μl of 2TY (1.6%tryptone, 1% yeast extract, 0.5% NaCl, pH 7.0)+100 μg/ml ampicillin+0.1%glucose media and incubated for 5 hours at 37° C., 280 rpm. 10 mM IPTGin TY was then added at 100 μl/well, and the block was incubatedovernight at 30° C., 280 rpm. In the morning, the block was spun down at3200 rpm for 15 minutes. For high-throughput screening, scFvsupernatants from the deep well block were transferred directly to theassay plate for the dilution required of 20%.

To test wells of a 384-well clear bottomed non-binding surface blackCostar plate the following were added: 10 μl sample (IgG or ScFv), 10 μldetection antibody or antibody mix, and 30 μl cells. Negative controlsused in these experiments typically involved addition of isotype (IgG)or irrelevant (ScFv) controls, or assay buffer in place of experimentalsample. The plates were sealed and incubated for four hours at roomtemperature in the dark and then read on the Applied Biosystems CellularDetection System 8200 reader. Data was typically analyzed with theVelocity algorithm, with gating set as color ratio <0.4, size 15-30, andmin count 20. Hits from the crude scFv supernatant samples were definedas showing 50% or greater inhibition of signal compared to the totalbinding control wells. Dose response curves were plotted for purifiedIgG titrations using Prism (Graphpad) curve-fitting software.

For IC₅₀ determinations, typically purified IgGs were diluted 2-fold inassay buffer from 500 nM to give 11 concentration points. 96-wellGreiner polypropylene (Greiner, 650201) plates were used for dilutionpreparation. Generally each dilution was prepared in duplicate.Alternatively, IgG testing was performed at a single concentration takenfrom the range 500 nM-0.2 nM.

Assessment of Antibody Binding on Cell Lines by Flow Cytometry

Binding of anti-ILT7 variants and isotype controls on human andcynomolgus ILT7 was assessed by flow cytometry analysis using CT-550 andcynoILT7 CT-125 cells respectively. CT-125 cells were used as control.Cells were resuspended in Blocking Buffer (PBS supplemented with 10%FBS) at a concentration of 5 million cells per mL and transferred into around-bottom 96-well plates (BD Falcon™ Clear Microtest Plate, BDBiosciences) at 100 μL per well. Anti-ILT7 variants and controlantibodies were added onto the cells for 30 min at 4° C. on a plateshaker. Cells were washed three times with PBS and resuspended inBlocking Buffer (100 μL/well). Human IgG binding on cell surface wasdetected using a secondary anti-human IgG antibody conjugated to DyLight649 (1 in 1000 dilution). Cells were incubated in the dark for 30 min at4° C. on a plate shaker. Cells were washed three times with PBS andsurface fluorescence was acquired using LSRII Flow Cytometry System andFACSDiva Software (both from BD Biosciences).

Assessment of Antibody Binding on Whole Blood and PBMCs by FlowCytometry

Binding of APC-labeled anti-ILT7 antibodies and isotype controls onhuman and cynomolgus whole blood was assessed by flow cytometryanalysis. Whole blood was transferred into 50 mL conical tubes, 1 mL pertube. APC-labeled antibodies were added directly into whole blood.Anti-BDCA-2-PE and anti-CD123-PE antibodies were used as plasmacytoiddendritic cell (pDC)-specific markers in human whole blood staining andcynomolgus whole blood staining, respectively. The whole blood wasincubated with the antibodies for 30 min at 4° C. in the dark on a plateshaker. Blood was treated with BD FACS Lysing Solution following themanufacturer's instructions. Cells were washed, and antibody binding wasassessed by flow cytometry using LSRII Flow Cytometry System andFACSDiva Software.

For PBMC staining, PBMCs were first washed with PBS and resuspended in acold PBS-based blocking buffer containing 50% human male AB plasma, 20μg/mL human IgG and 200 μL/mL of Human FcR Blocking Reagent for 15 minat 4° C. on a plate shaker. After 15 min, APC-labeled anti-ILT7 variantsor APC-labeled isotype control antibody were added directly into theblocking solution. Anti-BDCA-2-PE and anti-BDCA-4-PE antibodies werealternatively used as pDC-specific marker for human PBMC staining. Incynomolgus PBMCs, pDCs are defined as HLA-DR⁺, Lineage⁻, CD11c⁻ andCD123^(high) (Malleret et al., Immunology 124: 223-233 (2008)).Therefore, anti-HLA-DR PerCP-Cy5.5, Lineage-FITC (CD3, CD8, CD20 andCD14 antibodies) and anti-CD123-PE antibodies were used as apDC-specific marker for cynomolgus PBMC staining. PBMCs were incubatedfor 30 min 4° C. in the dark on a plate shaker. Cells were washed andantibody binding was assessed by flow cytometry using LSRII FlowCytometry System and FACSDiva Software.

Assessment of Antibody Potency by Antibody-Dependent Cell-MediatedCytotoxicity (ADCC) Assay Using Cell Lines

The potency of the anti-ILT7 antibodies was determined using an ADCC invitro cell-based assay. KC1333 cells (effectors) and CT cells (targets)were co-cultured at a 5:1 ratio (2.5×10⁵ KC1333 for 0.5×10⁵ CT cells) inround-bottom 96-well plates. Cells were co-cultured in presence ofanti-ILT7 antibodies or isotype control for 16 hours in RPMI 1640culture media supplemented with 10% FBS at 37° C., 5% CO₂. Cells werethen washed and transferred into blocking buffer (PBS-10% FBS). KC1333cells were detected using Pacific-Blue-anti-CD56 antibody. Dead cellswere detected using 7-AAD. Target cell viability was assessed by flowcytometry using LSRII Flow Cytometry System and FACSDiva Software. Thepercentage of cytotoxicity was obtained by applying the followingformula: percentage cytotoxicity=100−(number of live targets/number oflive target at baseline)×100.

Assessment of Antibody Potency by ADCC Assay Using Human PBMCs

Human PBMCs were washed with PBS and resuspended in RPMI mediasupplemented with 10% FBS and 200 ng/mL recombinant human IL-2 at aconcentration of 5.0×10⁶ cells per mL. PBMCs were seeded in duplicate inround bottom 96-well plates, 100 μl per well. 10-fold serial dilutionsof anti-ILT7 antibodies and control antibodies were prepared, and 100 μLof antibody solutions were added to appropriate wells for finalconcentrations of 33.85 nM-3.385 fM. Cells were incubated for 6 hours at37° C., 5% CO₂. Following incubations, cells were washed twice in 250 μLcold PBS. Cells were resuspended in 100 μL of cold PBS-based blockingbuffer containing 50% human male AB plasma, 20 μg/mL human IgG and 200μL/mL of Human FcR Blocking Reagent for 15 min at 4° C. Followingblocking step, 100 μL of cold blocking buffer containing FITC-anti-humanBDCA2 and APC-anti-human CD123 antibodies was added to appropriatewells. Plates were incubated, gently shaking for 30 minutes at 4° C.After incubation, cells were washed twice in 250 μL cold PBS with finalresuspension in 200 μL cold PBS. 50 μL of cold 7-AAD (Invitrogen)solution was added to all wells, and 7-AAD positive plasmacytoiddendritic cells were evaluated using LSRII Flow Cytometry System andFACSDiva Software.

IFNα Secretion Assays with Human PBMCs

Human PBMCs were washed with PBS and seeded in duplicate in roundbottom, 96-well plates at a final density of 150,000-156,000 cells perwell in RPMI media supplemented with 10% FBS and 200 ng/mL recombinanthuman IL-2. 10-fold serial dilutions of anti-ILT7 antibodies and controlantibodies were prepared, and 100 μL of antibody solutions were added toappropriate wells for final concentrations of 6.77 nM-0.677 fM. Cellsand antibodies were incubated for 9.5-10 hours at 37° C., 5% CO₂.Following incubations, 50 uL of ODN2216 (Invitrogen™) was added toappropriate wells for a final concentration of 0.5 μM, and plates werefurther incubated for an additional 16 hours at 37° C., 5% CO₂.Following incubations, plates were spun at 350 g for 10 minutes,supernatants were carefully removed, and IFNα was quantitated using amultisubtype IFNα ELISA kit (PBL Biomedical).

IFNα Secretion Assays with Cynomolgus Monkey PBMCs

Cynomolgus PBMCs were washed with PBS and resuspended in RPMI 1640supplemented with 10% FBS, 220 ng/mL recombinant human IL-2, and 500IU/mL recombinant human IFN-β. Maximum numbers of cells were added toappropriate wells with densities ranging from 314,000-818,000 cells perwell. 10-fold serial dilutions of anti-ILT7 antibodies and controlantibodies were prepared, and 100 μL of antibody solutions were added toappropriate wells for final concentrations of 33.85 nM-3.385 fM. Cellsand antibodies were incubated for 9.5-10 hours at 37° C., 5% CO₂.Following incubations, 50 μL of ODN2216 (Invitrogen™) was added toappropriate wells for a final concentration of 0.5 M, and plates werefurther incubated for an additional 16 hours at 37° C., 5% CO₂.Following incubations, plates were spun at 350 g for 10 minutes,supernatants were carefully removed, and supernatant IFNα wasquantitated using a rhesus/cynomolgus monkey IFNα ELISA kit (PBLBiomedical).

Statistical Analysis

EC₅₀ and IC₅₀ curves for binding, ADCC, and cytokine secretion assayswere generated using GraphPad Prims 5 software (GraphPad Software, CA,USA).

Example 1 Generation of Humanized ILT7 Antibodies from Murine AntibodySB128

Murine mAb SBI28 (SBI28 refers to the anti-ILT7 antibody ILT7#28provided in U.S. Published Application No. 2009/0280128) was humanizedby framework shuffling (Dall'Acqua et al., Methods 36:43-60 (2005)).Using this method, murine mAb SBI28 was humanized by synthesizing acombinatorial library comprised of its six CDRs fused in-frame to a poolof individual human germline frameworks. Human framework genes wereselected from the publicly available pool of antibody germline genes.These universal framework primer pools include 46 human germline kappachain genes, 5 human germline Jk sequences, 44 human germline heavychain genes, and 6 human germline JH sequences. Primer banks weredesigned to encode each framework of each germline gene.Antibody-specific CDR primers were also synthesized with degenerate endswhich overlap with the framework pools. The SBI28 framework shuffledlibrary was constructed by pairing the variable heavy chainframework-shuffled sub-library with the variable light chainframework-shuffled sub-library. The framework-shuffled sub-librarieswere assembled sequentially using the PCR by overlap extension. A firstfusion-PCR was carried out to synthesize each individual human germlineframework fused in-frame with a portion of the corresponding CDRs. Asecond “assembly-PCR” was then carried out using fusion-PCR product astemplate to amplify the full length VH and VL sub-libraries. The SBI28framework-shuffled library was cloned into a M13-based Fab expressionvector using Kunkel method hybridization mutagenesis. Approximately 1300clones from the SBI28 framework-shuffled library were screened on CHOcells expressing recombinant ILT7CHO-cell using the MesoScale Discovery(MSD) assay. One humanized variant, 10D10, bound with 3-fold loweraffinity to human ILT7 when compared to its chimeric parent (“SBI28ch”)as measured by surface Plasmon Resonance (SPR) on ProteOn. SBI28chrefers to the anti-ILT7 antibody ILT7#28 as provided in U.S. PublishedApplication No. 2009/0280128 which is herein incorporated by referencein its entirety.

Affinity optimization of 10D10 was initiated to improve its bindingaffinity to human and cynomolgus ILT7. 10D10 was first cloned into aM13-based ScFv expression vector for parsimonious mutagenesis. In thismethod, each individual amino acid of all 6 CDRs was randomly mutatedusing two separate libraries (NSS and NWS) per residue position. A totalof 12 independent libraries were constructed for 6 CDRs using Kunkelmethod hybridization mutagenesis. (Kunkel, T. A., et al. MethodsEnzymol. 154:367 (1987)) The screening of the synthesized librariesconsisted of a single-point ELISA designed to capture limitingconcentrations of secreted ScFv from bacterial culture media tonormalize the scFv concentration in each well. Labeled ILT7 antigenbound to the captured ScFv, and the signal strength of this interactioncorrelated with the relative binding affinity. Approximately 2,000 to3,000 clones were screened. To further engineer a variant with improvedaffinity, all beneficial single amino acid changes were encoded togethercreating a small and focused combinatorial library. In this step, 14individual positive hits at 9 positions in the 6 CDRs were encodedsimultaneously to build the combinatorial scFv library. Briefly,degenerate primers were designed that encoded all beneficial amino acidchanges as well as the parental residue at the same position. Thiscombinatorial library was screened with a single point capture ELISA aspreviously described. Approximately 1,200 clones were screened. Thevariable regions of the affinity-improved variant 7C7 was individuallycloned into the mammalian expression pOE vector and expressedtransiently in HEK293 cells. The secreted, soluble human IgGs werepurified directly from the conditioned media. Purified IgGs were assayedfor binding to rILT7 using ProteOn and FACS. In the ProteOn experiment,the affinity optimized antibody 7C7 showed about 60-fold KD improvementover SBI28ch. By FACs, where binding to recombinant human and cyno ILT7expressed on CHO cells was measured, 7C7 exhibited 2.2-fold and 14-foldbetter EC50 to human and cynomolgus ILT7, respectively, compared toSBI28ch. Alignments of the VH and VL sequences of SBI28, 10D10, and 7C7are provided in FIGS. 1A and 1B, respectively.

Example 2 Generation of Human ILT7 Antibodies from Human Library

In addition to humanizing a murine anti-ILT7 antibody (as describedabove in Example 1), human antibodies were generated using a library ofhuman sequences. Pursing multiple strategies for generating anti-ILT7antibodies maximizes the chances of generating anti-ILT7 antibodies withdistinct traits, so that the ideal antibody for a particular purpose canbe selected.

2.1 Selections

A large single chain Fv (scFv) human antibody library produced usingindividual heavy chain variable region and light chain variable regionsderived from bone marrow from adult naïve donors that were cloned into aphagemid vector based on filamentous phage M13 was used for selections(Hutchings, C., “Generation of Naïve Human Antibody Libraries” inAntibody Engineering, Dubel. Berlin, Springer Laboratory Manuals: p. 93(2001); Lloyd et al., Protein Eng. Des. Sel. 22(3):159-68 (2009)).ILT7-specific scFv antibodies were isolated from the phage displaylibrary in a series of repeated selection cycles on recombinant humanand/or cynomolgus ILT7 essentially as previously described in Vaughan etal. (Nat. Biotechnol. 14(3):309-14 (1996)). In brief, the scFv-phageparticles were incubated with biotinylated recombinant ILT7 in solution(biotinylated via free amines using EZ link Sulfo-NHS-LC-Biotin(Thermo/Pierce, product: 21335)). Typically, scFv-phage particles wereincubated with 100 nM biotinylated recombinant ILT7 for 1 hour. ScFvbound to antigen were then captured on streptavidin-coated paramagneticbeads (Dynabeads® M-280) following manufacturer's recommendations.Unbound phage was washed away in a series of wash cycles usingPBS-Tween. The phage particles retained on the antigen were eluted,infected into bacteria, and rescued for the next round of selection.Typically three rounds of selection were performed in this way.

2.2 Identification of ILT7 Specific Binders by Phage ELISA

scFvs were displayed on phage particles and tested in a binding assay todetermine cross-reactivity and specificity to recombinant antigens. Thedetailed assay method is provided in the Materials and Methods section.Approximately 2100 separate data points were generated from the bindingassay, and identified hits, i.e., scFv clones that showed binding torecombinant ILT7, were subjected to DNA sequencing (Osbourn et al.,Immunotechnology 2(3):181-96 (1996); Vaughan et al., Nat. Biotechnol.14(3):309-14 (1996)).

2.3 Identification of ILT7 Binders by FMAT

Unique scFvs were expressed in the bacterial periplasm and screened fortheir binding activity in a Fluorescence Microvolume Assay Technology(FMAT) binding assay. Binding of scFvs to ILT7 expressed on the cellsurface was detected using a goat anti-mouse Alexafluor-647 labeledantibody. The detailed assay method is provided in the Materials andMethods section.

2.4 Reformatting of scFv to IgG1

The most potent scFv binders were converted to whole immunoglobulin G1(IgG1) antibody format essentially as described by Persic et al (Gene187(1):9-18 (1997)) with the following modifications. An OriP fragmentwas included in the expression vectors to facilitate use withCHO-transient cells and to allow episomal replication. The VH domain wascloned into a vector (pEU1.3) containing the human heavy chain constantdomains and regulatory elements to express whole IgG1 heavy chain inmammalian cells. Similarly, the VL domain was cloned into a vector(pEU4.4) for the expression of the human light chain (lambda) constantdomains and regulatory elements to express whole IgG light chain inmammalian cells. To obtain IgGs, the heavy and light chain IgGexpressing vectors were transfected into CHO-transient mammalian cells.IgGs were expressed and secreted into the medium. Harvests were pooledand filtered prior to purification. Then IgG was purified using ProteinA chromatography. Culture supernatants were loaded on a column ofappropriate size of Ceramic Protein A (BioSepra) and washed with 50 mMTris-HCl pH 8.0, 250 mM NaCl. Bound IgG was eluted from the column using0.1 M Sodium Citrate (pH 3.0) and neutralized by the addition ofTris-HCl (pH 9.0). The eluted material was buffer exchanged into PBSusing Nap10 columns (Amersham, #17-0854-02), and the concentration ofIgG was determined spectrophotometrically using an extinctioncoefficient based on the amino acid sequence of the IgG (Mach et al.,Anal. Biochem. 200(1):74-80 (1992)).

2.5 Binding Assay for IgGs

Species cross-reactivity of anti-ILT7 antibodies was determined usingFMAT binding assay. The detailed assay method is provided in theMaterials and Methods section. The following 11 antibodies wereidentified as antibodies that successfully bound to both human andcynomolgus ILT7 in the FMAT screening assay: ILT70019, ILT70028,ILT70052, ILT70076, ILT70080, ILT70083, ILT70089, ILT70100, ILT70137,ILT70142, and ILT70144.

Example 3 ILT7 Antibodies Bind to ILT7-Expressing Cells

In order to determine the binding EC₅₀ of ILT70019, ILT70028, ILT70052,ILT70076, ILT70080, ILT70083, ILT70089, ILT70100, ILT70137, ILT70142 andILT70144 on cells expressing human ILT7, the candidates were screenedfor binding on CT-550 cells by flow cytometry. ILT70080 (EC₅₀=0.28 nM),ILT70083 (EC₅₀=0.37 nM), ILT70137 (EC₅₀=0.41 nM), ILT70144, ILT70142,ILT70052, and ILT70100 bound to human ILT7-expressing cells. CandidatesILT70019, ILT70028, and ILT70076 did not bind human ILT7-expressingcells. The anti-ILT7 antibodies 7C7 (7C7 is described above inExample 1) and SBI33 (SBI33 refers to the anti-ILT7 antibody ILT7#33provided in U.S. Published Application No. 2009/0280128) were used aspositive controls. Isotype control R347 was used as a negative controland did not show any binding on ILT7-expressing cells. The graph shownin FIG. 2 represents the mean results from two independent experiments,and the table shown in FIG. 2 shows the average EC₅₀.

In order to determine the binding EC₅₀ of the variants on cellsexpressing cynomolgus ILT7, the antibodies were screened for binding onCynoILT7 CT-125 cells by flow cytometry. ILT70052 (EC₅₀=0.35 nM),ILT70080 (EC₅₀=0.44 nM), ILT70083 (EC₅₀=1.37 nM), ILT70137 (EC₅₀=1.40nM), ILT70100 (EC₅₀=1.63 nM) and ILT70144 (EC₅₀=7.81 nM), ILT70142, andILT70089 were positive for binding on human ILT7. ILT70019, ILT70028,and ILT70076 did not bind cynomolgus ILT7-expressing cells. Isotypecontrol R347 did not show any binding on ILT7-expressing cells. Thegraph in FIG. 3 represents the mean results from two independentexperiments, and the table in FIG. 3 show the average EC₅₀.

Thus, all of ILT70052, ILT70080, ILT70083, ILT70100, ILT70137, ILT70142and ILT70144 bind to cells expressing either cynomolgus ILT7 or humanILT7. Particularly low EC₅₀ values were obtained with cells expressingboth cynomolgus and human ILT7 using, ILT70080, ILT70083, and ILT70137.

Example 4 ADCC Potency of ILT7 Antibodies

Anti-ILT7 antibodies were tested for ADCC potency against humanILT7-expressing cell lines using an in-vitro cell-based assay. Cellsexpressing human ILT7 (target cells) were plated in a proportion of 1:5with the natural killer (NK) cell line KC1333 (effector cells) in thepresence of anti-ILT7 variants or isotype control for 18 hours. Duringflow cytometry analysis, KC1333 cells were gated out using the NK markerCD56 (Biolegend #304624), and 7-AAD was used to distinguish viable fromdead cells. Using this method, the percentage of live target cells wascalculated and compared to the baseline (no antibody control).Cytotoxicity was calculated using the following formula:Percent cytotoxicity=100−(Number of live targets/Number of live targetsis no antibody control)×100

ILT70080 had the greatest ADCC potency against human ILT7-expressingcells (EC₅₀=0.022 nM), followed by ILT70137 (EC₅₀=0.044 nM) and ILT70083(EC₅₀=0.094 nM). ILT70142, ILT70052, ILT70100 and ILT70144 alsodisplayed ADCC activity (FIG. 4). Isotype control R347 and anafucosylated version of R347 (“Afuc R347”) did not display any ADCCactivity with human ILT7-expressing cells.

Anti-ILT7 antibodies were also tested for ADCC potency againstcynomolgus ILT7-expressing cells using the in-vitro cell-based activity.ILT70080 had the greatest ADCC potency against cynomolgusILT7-expressing cells (EC₅₀=0.008 nM), followed by ILT70137 (EC₅₀=0.015nM), ILT70142 (EC₅₀=0.058 nM), ILT70052 (EC₅₀=0.073 nM), ILT70144(EC₅₀=0.123), ILT70100 (EC₅₀=0.188 nM) and ILT70083 (EC₅₀=0.433 nM).ILT70089 also displayed ADCC activity. Positive control 7C7 displayedADCC, and isotype (negative) control R347 did not display any ADCC withcynomolgus ILT7-expressing cells. The graph and table in FIG. 5 arerepresentative of two independent experiments.

Thus, ILT70080 and ILT70137 showed the greatest ADCC activity in bothcynomolgus and human ILT-7 expressing cells.

Example 5 Binding of ILT7 Antibodies to PBMCs

The binding of anti-ILT7 antibodies ILT70080, ILT70083, and ILT70137 onhuman PBMCs was assessed by flow cytometry using an antibodyconcentration of 2.5 μg/ml. ITL70080, ILT70083, and ILT70137 bound tospecifically to pDCs (BDCA-4⁺ cells) (FIGS. 6A and B). Binding wasnegative with the isotype control R347.

The binding of anti-ILT7 antibodies ILT70080, ILT70083, and ILT70137 oncynomolgus PBMCs was also assessed by flow cytometry. ITL70080 andILT70083 bound specifically to pDCs (HLA-DR⁺, Lineage⁻, CD123^(high)cells).

Example 6 Effect of ILT7 Antibodies on IFN-Alpha Secretion

Anti-ILT7 variants were tested for ADCC potency in human and cynomolgusPBMCs as described above. IFNα secretion in the supernatant of PBMCscultured with anti-ILT7 antibodies and CpG-A was measured by ELISA.ILT70080, ILT70083, and ILT70137 all suppressed IFNα response to CpG-Ain human and cynomolgus PBMCs. ILT70080 had the greatest suppressiveeffect on the IFNα response.

Example 7 Afucosylation of ILT70080 and ILT70083 Antibodies

IgG1 antibodies contain two sites for N-linked oligosaccharides in theFc region, and these sites are heavily fucosylated in human antibodies.Antibody-dependent cellular cytotoxicity (ADCC) is mediated by thebinding of lymphocyte receptors to antibody Fc regions, and this isaffected by the amount of fucosylation. Increases in ADCC have beenobserved with decreased fucosylation. Therefore, afucosylated versionsof ILT7 were generated and analyzed.

7.1 Generation of the Afucosylated Version of Anti-ILT7 Antibodies

ILT70080 and ILT70083 IgG₁ were expressed in a CHO cell line that lacksthe enzyme α-1,6-fucosyltransferase. Expression in this cell lineresults in an antibody which lacks the α-1,6 fucose moiety on theN-glycan at Asn-297 of the heavy chain.

7.2 Testing of Afucosylated ILT70080 and ILT70083 Anti ILT7 Antibodies

A binding assay with afucosylated and parental ILT70080 and ILT70083antibodies on ILT7-expressing cells was performed to assess whetherafucosylation impacted the binding EC₅₀ of the antibodies. The parentaland the afucosylated antibodies showed similar binding on both human andcynomolgus ILT7-expressing cells (FIG. 7).

ADCC potency of afucosylated ILT70080 and ILT70083 antibodies was testedon human and cynomolgus ILT7-expressing cells using the in-vitrocell-based assay described above (Example 3). Afucosylation increasedADCC potency for all the candidates tested (FIG. 8). A ten-fold increasein potency was observed for ILT70080 antibody upon afucosylation in bothhuman and cynomolgus assays (from EC₅₀=0.013 nM to EC₅₀=0.001 nM, andfrom EC₅₀=0.006 nM to EC₅₀=0.00051 nM, respectively), while a 6 to7-fold increase was observed for ILT70083 (from EC₅₀=0.089 nM toEC₅₀=0.0105 nM, and from EC₅₀=0.36 nM to EC₅₀=0.057 nM, respectively).Afucosylated isotype control R347 did not display any ADCC withILT7-expressing cells.

The binding of afucosylated anti-ILT7 antibodies ILT70080 and ILT70083on human PBMCs was assessed by flow cytometry. Afucosylated variantsITL70080 and ILT70083 bound specifically to pDCs (BDCA-2⁺ cells).Binding was negative with the isotype control R347.

The binding of afucosylated anti-ILT7 variants ILT70080 and ILT70083 oncynomolgus PBMCs was also assessed by flow cytometry. Afucosylatedvariants ITL70080 and ILT70083 bound specifically to pDCs (HLA-DR⁺Lineage CD123^(high)). Binding was negative with the isotype controlR347.

Example 8 Engineering of ILT70080 and ILT70083 Antibodies

8.1 Engineering of ILT70080

The amino acid sequences of ILT70080 VH and VL were aligned to the knownhuman germline sequences in the VBASE database (Althaus H-H, Müller Wand Tomlinson I: V BASE; http://vbase.mrc-cpe.cam.ac.uk/), and theclosest germline sequence was identified by sequence similarity. For theVH domain this was VH1-69 (DP-10), and for the VL domain it wasVlambda3-h. Seven residues in the frameworks (FWs) of each of the VHdomain (A13K, T16S, L69I*, S70T, L80M, Y84S and D85E) and VL domain(E3V, K20R, S22T, M46L*, M48I*, F50Y* and I66N*) were selected forreversion to the closest germline sequence. The mutations marked with anasterisk are at positions classified as Vernier residues (Foote, J. etal. J. Mol. Biol. 224: 487 (1992)) and are typically left unchanged.However, from analysis of both Kabat (Wu, T. T. and Kabat E. A. J. Exp.Med. 132:211-250 (1970)) and IMGT (Lefranc, M.-P. et al. Dev. Comp.Immunol. 27: 55-77 (2003)) classification of CDRs, these positions wereconsidered to offer an additional opportunity to further reduceimmunogenicity with a low risk of altering the binding properties of theparent antibody. Additionally, a heavy chain N64Q mutagenesis wasperformed within the VH CDR2 (Kabat-defined) sequence, to remove apotential deamidation (NG) site at this position. Mutagenesis wasperformed on ILT70080 scFv sequence in pCantab6 (McCafferty et al., ApplBiochem Biotech 47:157 (1994)) using standard molecular biologytechniques. Different mutagenic oligonucleotide combinations wereutilized in multiple mutagenesis reactions to generate libraries ofsequences containing different combinations of FW mutations. Panels ofILT70080 scFv variants were then tested for retention of binding tohuman ILT7 as crude periplasmic extracts in an FMAT cell-binding assayas described above.

Seven ILT70080 variants were generated as IgG. See FIGS. 9A and 9B forVH and VL sequence alignments, respectively.

8.2 Engineering of ILT70083

Germlining of ILT70083 was also performed. The closest germlinesequences identified were VH3-23 (DP-47) and Vlambda1-b (DPL-5) for VHand VL sequences, respectively. One FW residue was selected formutagenesis in the VH domain (W66R), and eight FW residues were selectedin the VL domain, again including selected Vernier positions (V4L*,R42T, A64G*, I66K*, S68G*, A72T, A74G and E81G). ILT70083 variantscontaining different combinations of mutations were generated directlyon the pEU vectors containing separate VH and VL chains, using standardmolecular biology techniques. ILT70083 VH and VL chains were thenco-transfected in different combinations to generate nine ILT70083 IgG1variants. See FIGS. 10A and 10B for VH and VL alignments, respectively.

8.3 Testing of Engineered Antibodies

The resultant IgG1s were tested to confirm that the sequence changesincorporated into ILT70080 and ILT70083 and had not adversely impactedthe binding of the parent antibody to cells expressing human ILT7(CT-550 cells) or cynomolgus ILT7 (CT-125 cells). The variants werescreened for binding by flow cytometry. All ILT70080 variants had abinding similar to the parental ILT70080 antibody to human andcynomolgus ILT7 (EC₅₀=0.213 nM and 0.547 nM, respectively). See FIG. 11.The binding of ILT70083 variants was also similar the parental antibodyfor human ILT7 (EC₅₀=0.464 nM). See FIG. 12. However, five ILT70083variants (ILT70083.4, ILT70083.9, ILT70083.3, ILT70083.6, andILT70083.8) had improved binding capacity compared to the parentalantibody for cynomolgus ILT7. See FIG. 12.

Engineered ILT70080 and ILT70083 antibodies were tested for ADCC potencyagainst human ILT7-expressing cell lines using an in-vitro cell-basedassay. All ILT70080 variants had an increased ADCC potency compared tothe parental antibody (EC₅₀<14.1 pM). See FIG. 13. The two candidateswith the lowest EC₅₀ were ILT70080.6 (EC₅₀=6.9 pM) and ILT70080.1(EC₅₀=8.0 pM). The EC₅₀ values for the other ILT70080 variants were asfollows: ILT70080.1 EC₅₀=10.0 pM; ILT70080.3 EC₅₀=11.0 pM; ILT70080.4EC₅₀=11.9 pM; ILT70080.5 EC₅₀=8.6 pM; and ILT70080.7 EC₅₀=7.8 pM. AllILT70083 variants were found to have a decreased potency compared to theparental antibody (EC₅₀>89.0 pM). See FIG. 14.

Example 9 Afucosylation of Engineered ILT70080 and ILT70083 Antibodies

Anfucosylated version of ILT70080.6 was generated. Afucosylation of theILT70080.6 antibody did not affect its binding to either human orcynomolgus ILT7-expressing cells. The binding EC₅₀ of afucosylatedILT70080.6 on human and cynomolgus ILT7-expressing cells was 152.3 pMand 366.2 pM, respectively. See FIG. 15. The tables in FIG. 15 providethe mean results of three independent binding experiments measuring meanfluorescence intensity (MFI).

The ADCC activity of afucosylated versions of ILT70080.6 and ILT70083(see Example 7 above) was also assessed. Afucosylation of ILT70080.6improved its ADCC potency against both human and cynomolgusILT7-expressing cells by about 10-fold. See FIG. 16. The EC₅₀ ofafucosylated ILT0080.6 was 1.12 pM against human ILT7-expressing cellsand 0.44 pM against cynomolgus ILT7-expressing cells. The tables in FIG.16 provide the mean results of three independent ADCC assays measuringcytotoxicity.

Afucosylated ILT70080.6 and ILT70083 were tested for ADCC potency inhuman PBMCs. Cytotoxicity of the antibodies was assessed by flowcytometry and CpG A-mediated IFNα secretion in the supernatant wasmeasured by ELISA. The results are shown in FIG. 17. In cynomolgusPBMCs, the EC50 values for IFNα secretion using afucosylated ILT70080.6and ILT70083 antibodies were 58 pM and 5216 pM, respectively.

In human whole blood and PBMCs, afucosylated ILT70080.6 and ILT70083antibodies were found to bind specifically to BDCA-2 positive cells.Binding of both antibodies was restricted to human pDC at allconcentration tested (0.1-5.0 μg/mL).

In cynomolgus whole blood, afucosylated ILT70080.6 and ILT70083antibodies were found to bind to pDCs (HLA-DR⁺ Lineage⁻ CD123^(high)cells) at all concentrations tested (0.5-2.5 μg/ml).

Example 10 Afucosylation of ILT70137 Antibody

An afucosylated version of the ILT70137 antibody was made as describedabove in Example 7 for the ILT70080 and ILT70083 antibodies.

10.1 Binding to Soluble Recombinant Human ILT7

BIAcore (surface plasmon resonance) was used to measure the kinetic rate(k_(on), k_(off)) constants for the binding of afucosylated IgG1ILT70137 to human ILT7 protein using an IgG-capture assay format. Thebinding of each concentration of a two-fold dilution series of the ILT7protein was recorded after first capturing IgG onto the sensor chipsurface, followed by either the ILT7 protein or instrument buffer. Inbetween each pair of injections, the IgG capture surface wasregenerated. Individual association and dissociation rate constants werethen calculated from the resulting binding curves using theBiaevaluation software available through the vendor's software employinga 1:1 fitting model, which included a term to correct for mass transportlimited binding, should it be detected. From a high-resolution BIAcoreplot of the data, the association rate constant and dissociation rateconstants for the binding of ILT7 protein to afucosylated IgG₁ ILT70137was determined to be 1.855×10⁵ M⁻¹ s⁻¹. This same plot was also used todetermine the corresponding dissociation rate constant for thisinteraction, which measured 3.175×10⁻² s⁻¹. From these rate constants,the K_(D) was then calculated from the quotient of k_(off)/k_(on) to be171 nM. These results are summarized in Table 3, below. The individualerrors for k_(on) and k_(off) were low, and the overall fit to the datawas good as judged by Chi2 values of ˜1% of the calculated R. (maximumresponse). Taken together, this suggests that the use of the one-sitebinding model to fit the data was appropriate. The evaluation did notindicate the binding was mass transport limited, so the measuredassociation rate constants are considered valid.

TABLE 3 Summary of Kinetic Rate Constant and KD Data for the Binding ofHuman ILT7 Protein to Afucosylated IgG₁ ILT70137 Interaction k_(on)(×10⁵ M⁻¹s⁻¹) k_(off) (×10⁻² s⁻¹) K_(D) (nM) huILT7/Afuc IgG₁ 1.8553.175 171 ILT70137 K_(on) = association rate constant; K_(off) =dissociation rate constant; K_(D) = equilibrium binding constant.10.2 Binding to ILT7-Expressing Cell Lines

Binding of afucosylated ILT70137 to ILT7 was determined using cell linesstably expressing human or cynomolgus monkey ILT7. The mean fluorescenceintensity of cell-bound antibodies was assessed by flow cytometry. Cellswere incubated with increasing concentrations of test antibody rangingfrom 0.004 to 333.3 nM for 30 minutes at 4° C. After incubation, cellswere washed with cold PBS and incubated for 30 minutes at 4° C. with ananti-human-Alexa Fluor 647 antibody. Fluorescent intensity was thendetermined by FACS and EC₅₀ values were calculated using a non-linearfit equation in GraphPad Prism 6 software.

The results are shown in FIG. 18. Afucosylated ILT70137 was found tobind recombinant human and cynomolgus monkey ILT7-expressing cells in adose-dependent manner. No significant binding was observed with isotypecontrols. The average afucosylated ILT70137 half maximal effectiveconcentration (EC₅₀) was 0.303 nM for binding to human ILT7-expressingand 2.148 nM for cynomolgus monkey ILT7-expressing cells.

10.3 ADCC Activity on ILT7-Expressing Cell Lines

The potential of afucosylated ILT70137 to induce ADCC was measured by afluorescence-activated cell sorting (FACS) assay on target cells thatexpress human or cynomolgus monkey ILT7. Target cells were co-culturedwith effector NK cells line KC1333 at a ratio of 1:5 in the presence ofincreasing concentrations of afucosylated ILT70137 or an isotype control(ranging from 0-6.66×10⁻⁹ M). For assessment of target cell viability byflow cytometry, KC1333 were gated out using CD56, and dead cells weregated out using 7-amino-actinomycin D (7-AAD) viability stain. Theviable target cells were defined as CD56 negative, 7-AAD negative. Thepercentage of cytotoxicity was calculated using the following formula: %Cytotoxicity=100-(percentage of live target cells/percentage of livetargets in No Antibody controls)×100. Half-maximal effectiveconcentration values (EC₅₀) values were calculated using a non-linearfit equation in GraphPad Prism 6 software. x-axis: antibodyconcentration.

The results are shown in FIG. 19. Afucosylated ILT70137 induced ADCC oncells expressing ILT7 in a dose-dependent manner with an EC₅₀ of 4.19 pMon cells expressing human ILT-7 and 1.89 pM for cells expressingcynomolgus monkey ILT7.

10.4 ADCC Activity on Primary Plasmacytoid Dendritic Cells

IFN-α secretion in response to a Toll-like receptor 9 (TLR9) agonist islargely due to plasmacytoid dendritic cells (pDCs) in the peripheralblood mononuclear cell (PBMC) preparation. Therefore, the potential ofafucosylated ILT70137 to induce ADCC of primary pDCs was indirectlymeasured by assessing its ability to block the secretion of IFN-α inPBMCs. In these assays, purified PBMCs were plated in 96-well, roundbottom plates in media supplemented with 10% fetal bovine serum and 200ng/mL recombinant human IL-2. Serial dilutions of afucosylated ILT70137and the control antibodies were added to appropriate wells in duplicateand incubated for 9.5 hours. After incubation, 50 μL of the TLR9 agonistODN2216 was added to each well for a final concentration of 0.5 μM.IFN-α was quantitated in supernatants using a multisubtype IFN-α ELISAkit, and is presented as pg/mL of supernatant in FIG. 20. The IC50 ofADCC was calculated using a non-linear fit equation in GraphPad Prismv5.01 software.

Afucosylated ILT70137 reduced TLR9-mediated secretion of IFN-α in PBMCsin a dose-dependent manner with a half-maximal inhibitory concentration(IC₅₀) of 0.048 nM. These results indicate that afucosylated ILT70137effectively depletes naturally occurring primary human pDCs in PBMCs.

10.5 Binding to Primary Plasmacytoid Dendritic Cells

The specificity of afucosylated ILT70137 to human primary plasmacytoiddendritic cells (pDCs) was assessed by FACS in peripheral bloodmononuclear cells (PBMCs). PBMCs were isolated from human donors. Inorder to properly identify this dendritic cell subset, the markers CD123(expressed on pDCs and basophils) and CD304 (unique to pDCs) were firstutilized. The pDCs were CD123+CD304+ double positive, and CD304 stainingwas sufficient to identify pDCs. See FIG. 21 (upper panel). AfucosylatedILT70137 bound only to CD304 positive cells, indicating that it bindsuniquely to pDCs. See FIG. 21 (lower right panel). No significantbinding to this population was observed with the human IgG1 isotypeafucosylated control antibody R3-47. See FIG. 21 (lower left panel).

Example 11 In Vivo Activity of ILT7 Antibodies

Three anti-ILT7 antibodies were administered to male cynomolgus monkeys:afucosylated 7C7, afucosylated ILT70080.6, and afucosylated IgG1ILT70137. All three antibodies were active in depleting plasmacytoiddendritic cells (pDCs).

Administration of afucosylated ILT70080.6 was generally well tolerated.However, the following pathological findings were observed: decreasedneutrophil count, vascular leukocytosis, an increase in glomerularmatrix, and vascular/perivascular inflammation. In addition, theappearance of antibodies against the afucosylated ILT70080.6 antibody(anti-drug antibodies) was associated with increased clearance of theafucosylated ILT70080.6.

In another study, the toxicokinetics of afucosylated 7C7 andafucosylated ILT70137 were studied. In this study 5 equivalent doses ofthe antibodies were administered to cynomolgus monkeys by infusion.Following administration, exposure was comparable between afucosylated7C7 and afucosylated ILT70137 at steady state. In addition, as shown inFIG. 22, a specific and reversible depletion of pDCs was achieved usingeither antibody. The pDC depletion lead to an ex-vivo inhibition of IFNαproduction. See FIG. 23.

However, the pathology of animals treated with afucosylated 7C7 andafucosylated ILT70137 was different. Increased spleen weights wereobserved in some animals treated with afucosylated 7C7. Microscopicfindings were also observed in some animals treated with afucosylated7C7. In particular, red pulp and macrophage hypertrophy/hyperplasia wereobserved in spleens. Kupffer cell hypertrophy/hyperplasia was observedin livers. In addition, immunohistochemistry showed human IgG/7C7 andmonkey IgG-containing granular deposits associated withhypertrophied/hyperplastic Kupffer cells in liver and red pupmacrophages in spleen. These observations are consistent withexaggerated physiological clearance of immune complexes containing drug(7C7) and anti-drug antibodies (ADA). In contrast, no changes in organweights and no macroscopic or microscopic findings were observed withafucosylated afucosylated ILT70137.

In addition, while neutrophil counts dropped below 1 E3/μl for twomonkeys treated with the afucosylated 7C7 antibody, no significantchanges in neutrophil count were observed in monkeys treated withcontrol or afucosylated afucosylated ILT70137.

Thus, while all three antibodies depleted pDCs in vivo, the superiorsafety and lack of anti-drug antibodies after administration ofafucosylated afucosylated ILT70137 is surprisingly advantageous.

Example 12 Epitope Mapping

In order to determine the epitope bound by the ILT7 antibodies, chimericpolypeptides containing ILT7 and ILT1 polypeptides were constructed, andthe binding of ILT7 antibodies to these constructs was tested. ILT1(Accession Number Q8N149) was selected to construct chimeric variantsbecause it has the same modular structure as ILT7 and shares 65%identity with ILT7, but is not recognized by ILT7 monoclonal antibodies.The chimeric polypeptides were generated by replacing the extracellularIg domains of ILT7 with ILT1 counterparts. All of these constructscontained an N-terminal Flag tag. The results demonstrated that ILT70080and ILT70083 bind to the Ig1 domain of ILT7. In contrast, the 7C7antibody binds to the Ig2 domain of ILT7.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingknowledge within the skill of the art, readily modify and/or adapt forvarious applications such specific embodiments, without undueexperimentation, without departing from the general concept of thepresent invention. Therefore, such adaptations and modifications areintended to be within the meaning and range of equivalents of thedisclosed embodiments, based on the teaching and guidance presentedherein. It is to be understood that the phraseology or terminologyherein is for the purpose of description and not of limitation, suchthat the terminology or phraseology of the present specification is tobe interpreted by the skilled artisan in light of the teachings andguidance.

The breadth and scope of the present invention should not be limited byany of the above-described exemplary embodiments, but should be definedonly in accordance with the following claims and their equivalents.

What is claimed is:
 1. An anti-Immunoglobulin-like transcript-7 (ILT7)antibody comprising Complementarity-Determining Regions (CDRs) HCDR1,HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein the HCDR1, HCDR2, HCDR3,LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ IDNOs:203, 204, 205, 208, 209, and 210, respectively.
 2. The anti-ILT7antibody of claim 1, wherein the antibody comprises a heavy chainvariable region (VH) and a light chain variable region (VL), wherein theVH and VL regions comprise an amino acid sequence at least 95% identicalto SEQ ID NO:202 and SEQ ID NO:207, respectively.
 3. The anti-ILT7antibody of claim 2, wherein the VH and VL regions comprise the aminoacid sequence of SEQ ID NO:202 and SEQ ID NO:207, respectively.
 4. Theanti-ILT7 antibody according to claim 1, wherein the antibody comprisesa heavy chain immunoglobulin constant domain selected from the groupconsisting of an IgA constant domain, an IgD constant domain, an IgEconstant domain, an IgG1 constant domain, an IgG2 constant domain, anIgG3 constant domain, an IgG4 constant domain, and an IgM constantdomain.
 5. The anti-ILT7 antibody of claim 4, wherein the antibodycomprises an IgG1 constant domain.
 6. The anti-ILT7 antibody accordingto claim 1, wherein the antibody comprises a light chain immunoglobulinconstant domain selected from the group consisting of an Ig kappaconstant domain and an Ig lambda constant domain.
 7. The anti-ILT7antibody of claim 6, wherein the antibody comprises a lambda constantdomain.
 8. The anti-ILT7 antibody according to claim 1, wherein theantibody is a monoclonal antibody.
 9. The anti-ILT7 antibody accordingto claim 1, wherein the antibody is a human, chimeric, or resurfacedantibody.
 10. The anti-ILT7 antibody of claim 9, wherein the antibody isa human antibody.
 11. The anti-ILT7 antibody according to claim 1,wherein the antibody is afucosylated.
 12. The anti-ILT7 antibodyaccording to claim 1, wherein the antibody specifically binds to humanand cynomolgus ILT7.
 13. An afucosylated, human, monoclonal anti-ILT7antibody comprising: i. Complementarity-Determining Regions (CDRs)HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3; ii. a human IgG1 constantdomain; and iii. a human lambda constant domain, wherein the HCDR1,HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequencesof SEQ ID NOs:203, 204, 205, 208, 209, and 210, respectively.
 14. Apolynucleotide comprising a nucleic acid encoding the anti-ILT7 antibodyof claim
 1. 15. A isolated host cell comprising the polynucleotide ofclaim
 14. 16. A method of producing an anti-ILT7 antibody, comprisingculturing the isolated host cell of claim 15, and recovering saidantibody.
 17. A method for detecting ILT7 expression in a samplecomprising (a) contacting said sample with the anti-ILT7 antibody ofclaim 1 and (b) detecting binding of said antibody in said sample.
 18. Amethod for decreasing interferon (IFN)-alpha release from a plasmacytoiddendritic cell (pDC), comprising contacting a pDC with the anti-ILT7antibody of claim
 1. 19. A method for treating a human subject with anautoimmune disease comprising administering to the subject an effectiveamount the anti-ILT7 antibody of claim
 1. 20. The method of claim 19,wherein the autoimmune disease is selected from the group consisting of:myositis, diabetes, Hashimoto's disease, autoimmune adrenalinsufficiency, pure red cell anemia, multiple sclerosis, rheumatoidcarditis, systemic lupus erythematosus, psoriasis, rheumatoid arthritis,chronic inflammation, Sjogren's syndrome, polymyositis, dermatomyositis,inclusion body myositis, juvenile myositis, and scleroderma.
 21. Themethod of claim 20, wherein the autoimmune disease is systemic lupuserythematosus.
 22. An afucosylated IgG1 human monoclonal anti-ILT7antibody comprising a heavy chain variable region (VH) and a light chainvariable region (VL), wherein the heavy chain variable region comprisesthe amino acid of SEQ ID NO:202 and the light chain variable regioncomprises the amino acid of SEQ ID NO:207.
 23. The anti-ILT7 antibody ofclaim 22, wherein the antibody comprises a human IgG1 constant domain;and a human lambda constant domain.
 24. A pharmaceutical compositioncomprising the afucosylated IgG1 human monoclonal anti-ILT7 antibody ofclaim 22.